Molecules that selectively home to vasculature of pre-malignant dysplastic lesions or malignancies

ABSTRACT

The present invention provides a conjugate that contains a therapeutic moiety linked to a homing peptide or peptidomimetic which selectively homes to vasculature of pre-malignant dysplastic skin and which includes the amino acid sequence SRPRR (SEQ ID NO: 1) or a conservative variant or peptidomimetic thereof. The present invention further provides a conjugate containing a therapeutic moiety linked to a homing peptide or peptidomimetic which selectively homes to vasculature of malignant skin and which includes the amino acid sequence CGKRK (SEQ ID NO: 6) or the amino acid sequence CDTRL (SEQ ID NO: 7), or a conservative variant or peptidomimetic of one of these sequences.

This application claims benefit of provisional application Ser. No.60/513,407, filed Oct. 21, 2003, which is herein incorporated byreference.

This invention was made with government support under CA 82713, CA30199, and CA 37395 awarded by the National Cancer Institute. Thegovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cancer andcancer progression, molecular medicine and drug delivery and, morespecifically, to molecules that selectively home to vasculature ofpre-malignant dysplastic lesions or to vasculature of malignancies.

2. Background Information

A major hurdle to advances in preventing or treating cancer is the lackof agents that are effective in selectively targeting a cancer orpre-cancerous tissue while sparing normal cells. Radiation therapy andsurgery, for example, which generally are localized treatments, cancause substantial damage to normal tissue in the treatment field,resulting in scarring and loss of normal tissue. Furthermore,chemotherapy, which generally is administered systemically, can causesubstantial damage to normal organs such as normal skin, bone marrow,mucosa, and small intestine, which undergo rapid cell turnover andcontinuous cell division. As a result, undesirable side effects such asnausea, loss of hair and drop in blood cell count occur as a result ofsystemic treatment with a chemotherapeutic agent. Such undesirable sideeffects often limit the amount of drug that can be safely administered,thereby reducing survival rate and impacting the quality of patientlife.

Selective delivery of therapeutics such as anti-angiogenic agents tovasculature that supports tumors would result in less toxic therapysince rapidly proliferating normal cells would be spared. Similarly,selective delivery of anti-angiogenic agents to vasculature ofdysplastic cells that are not yet malignant would provide a prophylacticstrategy for reducing the risk of cancer. However, to date, it has beendifficult to produce drugs that are delivered specifically to tumorvasculature or the vasculature of dysplastic, pre-malignant tissues.Thus, there is a need for molecules that selectively target malignanttissue such as malignant skin, as well as for molecules that selectivelytarget pre-malignant dysplastic tissues such as pre-malignant dysplasticskin. The present invention satisfies these needs and also providesrelated advantages.

SUMMARY OF THE INVENTION

The present invention provides a conjugate that contains a therapeuticmoiety linked to a homing peptide or peptidomimetic which selectivelyhomes to vasculature of pre-malignant dysplastic skin and which includesthe amino acid sequence SRPRR (SEQ ID NO: 1) or a conservative variantor peptidomimetic thereof.

The present invention also provides a method of directing a moiety tovasculature of a pre-malignant dysplastic tissue in a subject byadministering to the subject a conjugate containing a moiety linked to ahoming molecule which selectively homes to vasculature of pre-malignantdysplastic skin and which specifically binds a cognate receptor forCSRPRRSEC (SEQ ID NO: 3), thereby directing the moiety to vasculature ofthe pre-malignant dysplastic tissue.

Further provided herein is a method of imaging vasculature of apre-malignant dysplastic tissue by administering to the subject aconjugate containing a detectable moiety linked to a homing moleculewhich selectively homes to vasculature of pre-malignant dysplastic skinand which specifically binds a cognate receptor for CSRPRRSEC (SEQ IDNO: 3); and detecting the conjugate, thereby imaging vasculature of thepre-malignant dysplastic tissue.

The present invention also provides a method of reducing the risk ofprogression to a malignancy in a subject by administering to the subjecta conjugate containing a therapeutic moiety linked to a homing moleculewhich selectively homes to vasculature of pre-malignant dysplastic skinand which specifically binds a cognate receptor for CSRPRRSEC (SEQ IDNO: 3), thereby diminishing vasculature of a pre-malignant dysplastictissue and reducing the risk of progression to the malignancy.

Also provided herein is a conjugate containing a therapeutic moietylinked to a homing peptide or peptidomimetic which selectively homes tovasculature of malignant skin and which includes the amino acid sequenceCGKRK (SEQ ID NO: 6) or the amino acid sequence CDTRL (SEQ ID NO: 7), ora conservative variant or peptidomimetic of one of these sequences.

The present invention further provides a method of directing a moiety tovasculature of a malignant tissue in a subject by administering to thesubject a conjugate containing a moiety linked to a homing moleculewhich selectively homes to vasculature of malignant skin and whichspecifically binds a cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7,thereby directing the moiety to vasculature of the malignant tissue.

Additionally provided by the present invention is a method of imagingvasculature of a malignant tissue by administering to the subject aconjugate containing a detectable moiety linked to a homing moleculewhich selectively homes to vasculature of malignant skin and whichspecifically binds a cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7;and detecting the conjugate, thereby imaging vasculature of themalignant tissue.

The present invention also provides a method of treating a cancer in asubject by administering to the subject a conjugate containing atherapeutic moiety linked to a homing molecule which selectively homesto vasculature of malignant skin and which specifically binds a cognatereceptor for SEQ ID NO: 6 or SEQ ID NO: 7, thereby directing thetherapeutic moiety to vasculature of the cancer and treating the cancer.

Further provided by the present invention is a method of staging tumorprogression in a subject having or suspected of having a pre-malignantlesion or tumor by administering to the subject a conjugate containing adetectable moiety linked to (i) a homing molecule which selectivelyhomes to vasculature of pre-malignant dysplastic skin and whichspecifically binds a cognate receptor for CSRPRRSEC (SEQ ID NO: 3), or(ii) a homing molecule which selectively homes to vasculature ofmalignant skin and which specifically binds a cognate receptor for SEQID NO: 6 or SEQ ID NO: 7; and detecting the conjugate, where detectionof the conjugate containing a homing molecule which selectively homes tovasculature of pre-malignant dysplastic skin indicates a pre-malignantstage of tumor progression in the subject and where detection of theconjugate containing a homing molecule which selectively homes tovasculature of malignant skin indicates a malignant stage of tumorprogression in the subject.

The present invention further provides an isolated peptide orpeptidomimetic which has a length of less than 20 residues and includesthe amino acid sequence SRPRR (SEQ ID NO: 1) or a peptidomimeticthereof. Also provided herein is an isolated peptide or peptidomimeticwhich has a length of less than 90 residues and which includes the aminoacid sequence CXSRPRRZC (SEQ ID NO: 2) or a peptidomimetic thereof,where X=0 to 20 independently selected residues and Z=0 to 20independently selected residues.

In addition, there is provided by the present invention an isolatedpeptide or peptidomimetic which has a length of less than 20 residuesand which includes the amino acid sequence CGKRK (SEQ ID NO: 6) or apeptidomimetic thereof. Further provided herein is an isolated peptideor peptidomimetic which has a length of less than 20 residues and whichincludes the amino acid sequence CDTRL (SEQ ID NO: 7) or apeptidomimetic thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that phage-displayed peptides selectively home todysplastic skin lesions and tumors. Ex vivo and in vivo selections ofphage were performed for binding and homing to (A) dysplastic skinlesions or (B) skin tumors. (C) In vivo homing of CSRPRRSEC (SEQ ID NO:3) phage from the dysplastic skin screen to dysplastic skin lesions of a4 to 6 month-old K14HPV16 mouse (left) and to dysplastic skin lesionsand tumor of a 9 to 12 month-old K14HPV16 mouse (right). In vivo homingof (D) CGKRK (SEQ ID NO: 6) and (E) CDTRL (SEQ ID NO: 7) peptides fromtumor screening to tumors and other tissues in K14HPV16 mice. Resultsfrom two different tumor-bearing mice are shown for each peptide. Normalskin values shown in panels C to E are from parallel experiments inwild-type FVB/n mice.

FIG. 2 shows vascular localization of homing phage. K14HPV16 mice withdysplastic skin lesions or tumors were intravenously injected withindividual homing phage before sacrificing mice 10 minutes later, anddetecting phage in tissue sections with rabbit anti-T7 phage antisera(Alexa594). Blood vessels were stained with rat anti-mouse CD31(Alexa488). (A) CSRPRRSEC (SEQ ID NO: 3)-displaying phage co-localizewith CD31 in the dysplastic skin lesions of 4-6 month-old dysplasticmice. (B and C) Tumor-homing CGKRK (SEQ ID NO: 6)-phage home toCD31-positive vessels in dysplasias and skin tumors. (D and E)Tumor-homing CDTRL (SEQ ID NO: 7)-phage phage home to CD31-positivevessels in dysplasias and skin tumors. Magnifications shown are 200×.

FIG. 3 shows that fluorescein-labeled peptides co-localize with avascular marker. Fluorescein-labeled peptides were intravenouslyinjected into mice with dysplastic skin lesions or tumors beforesacrificing mice 10 minutes later and analyzing peptide localization intissues sections. Fluorescein-CSRPRRSEC (SEQ ID NO: 3) co-localizes withMeca-32 in the vasculature of dysplastic skin (A) but not in tumortissue (D). The CSRPRRSEC (SEQ ID NO: 3) peptide continues to recognizethe vasculature of dysplastic skin in tumor-bearing mice (D inset) butdoes not recognize pre-malignant lesions (angiogenic islets) inRIP1-Tag2 mice. Fluorescein-labeled CGKRK (SEQ ID NO: 6) and CDTRL (SEQID NO: 7) peptides were not detected in dysplastic skin from 4-6month-old mice (B and C), but co-localized with Meca-32 in tumorvasculature (E and F). Magnifications shown are 200×.

FIG. 4 shows the localization of fluorescein-labeled peptides in varioustumors. Mice bearing various tumors were intravenously injected withfluorescein-labeled peptides and examined as in FIG. 3. Tissue sectionswere stained with Meca-32 and CD31. Fluorescein-CGKRK (SEQ ID NO: 6)(A-E) was detected in four of the five tumor models examined; RIP1-Tag2tumors were negative as shown in (A). The CGKRK (SEQ ID NO: 6) peptidewas seen in endothelial cells and tumor cells and appeared in both thecytoplasm and nucleus (B-E). Fluorescein-CDTRL (SEQ ID NO: 7) waspresent in MMTV-PyMT tumors (G) and C8161 xenografts (J). In the C8161xenografts, the positive cells were CD31-positive cells that wereadhering and spreading on the lumenal surface of the blood endothelialcells; the established endothelia were negative (J). Blood vessels inthe skin surrounding the C8161 xenograft tumor are also positive forfluorescein-CDTRL (SEQ ID NO: 7) (J inset). In MMTV-PyMT mice,fluorescein-CDTRL (SEQ ID NO: 7) co-localized with CD31 and Meca-32 (G),and also bound to tumor cells within the vessel wall (G). Magnificationsshown are 200×.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to molecules that selectively home tovasculature of pre-malignant dysplastic skin and further directed tomolecules that selectively home to vasculature of malignant skin. Asdisclosed herein in Example I, peptides specific for pre-malignant,dysplastic lesions were isolated using a combination of in vivo and exvivo selections on 4 to 6 month old K14-HPV16 mice with dysplastic skinlesions but no macroscopic evidence of tumors. Sequential ex vivoselections using suspensions of dysplastic skin cells resulted in a160-fold enrichment of phage relative to enrichment of non-recombinantphage lacking displayed peptides (FIG. 1A, left); greater than10,000-fold enrichment resulted from the subsequent round of in vivoselection (FIG. 1A, right).

To test homing specificity of selected peptides, purified phagedisplaying a single recombinant sequence were intravenously injectedinto K14HPV16 mice bearing protuberant ear or trunk tumors at 9 to 12months of age to assess homing to squamous cell carcinomas, oralternatively injected into younger K14HPV16 mice presenting withmultifocal dysplasias but no tumors. Both neoplastic tissues and normalcontrol organs were collected and assayed for phage accumulation.

Peptides displayed on several of the selected phage clones were found tobe highly selective for dysplastic skin, and did not appreciably home tonormal organs. One of these dysplasia-homing peptides, CSRPRRSEC (SEQ IDNO: 3), appeared three times amongst the 48 phage sequenced from the invivo round, along with two variants, CSRPRRSVC (SEQ ID NO: 4) andCSRPRRSWC (SEQ ID NO: 5), that each appeared once. As disclosed hereinin FIG. 1C, left panel, phage displaying peptide CSRPRRSEC (SEQ ID NO:3) were enriched ˜350-fold in dysplastic skin and did not significantlyaccumulate in control tissues. Furthermore, when injected into aK14HPV16 mouse bearing an ear tumor and multifocal skin dysplasias,CSRPRRSEC (SEQ ID NO: 3)-displaying phage effectively homed todysplastic chest skin and dysplastic ear skin with little homing to thetumor (see FIG. 1C, right panel). In addition, CSRPRRSEC (SEQ ID NO:3)-bearing phage did not home to normal skin of FVB/n mice in vivo andfurther did not bind to hyperplastic skin of 1 to 2 month-old K14-HPV16mice in ex vivo experiments (FIG. 1C, left and right panels). Resultsdisclosed herein further indicate that the dysplasia-homing sequence,CSRPRRSEC (SEQ ID NO: 3), is homologous to the C₂₂₀SRPRR₂₂₅ (SEQ ID NO:17) loop that defines substrate specificity in human kallikrein 9 (humanKLK-9), a protease of the trypsin super-family which is a positiveprognostic marker in breast and ovarian cancer. Thus, a peptidemimicking the active site loop of human kallikrein 9 binds to bloodvessels of pre-malignant or angiogenic dysplasias but not to bloodvessels associated with a malignancy.

These results demonstrate that peptide CSRPRRSEC (SEQ ID NO: 3)selectively homes to dysplastic skin lesions and further indicate thatthis peptide binds to a receptor which is present in skin dysplasias butwhich is essentially absent or inaccessible via the circulation innormal skin and in squamous cell carcinomas. In view of the localizationof this peptide to endothelial cells, these results further indicatethat homing selectivity of peptide SEQ ID NO: 3 is attributable tovascular changes during the carcinogenic progression from normal skin todysplasia and from dysplasia to cancer.

As further disclosed herein in Example II, phage that selectively hometo squamous cell carcinomas (SCCs) were isolated by two rounds of exvivo panning followed by two rounds of in vivo panning in K14-HPV16 micehaving tumors histologically confirmed as squamous cell carcinoma gradesII-IV (Coussens et al., Am. J. Pathol. 149:1899-1917 (1996)). As shownin FIG. 1B, phage enrichment relative to non-recombinant phage rose from6-fold in the second ex vivo round to greater than 70-fold relative tonon-recombinant phage in the second in vivo round, which was the fourthsequential round overall. After sequencing of almost 200 phage clones,several were chosen for further analysis based on their frequency andincreased prevalence in in vivo selections. Of the fifteen chosenclones, phage displaying CGKRK (SEQ ID NO: 6), CGTKRKC (SEQ ID NO: 11),CDTAVVEGL (SEQ ID NO: 12) or CDTRL (SEQ ID NO: 7) bound to a K14-HPV16tumor-derived cell suspension ex vivo.

Furthermore, upon intravenous injection into tumor-bearing K14-HPV16mice, CGKRK (SEQ ID NO: 6) phage showed a marked preference for tumors.Similar analysis of the CDTRL (SEQ ID NO: 7) phage revealed a variablepreference for squamous cell carcinomas and dysplastic lesions; in oneexperiment the phage accumulated more effectively in dysplastic lesionsthan in a tumor, whereas the reverse was true in another experiment,indicative of lesional heterogeneity in the CDTRL (SEQ ID NO: 7) cognatereceptor (FIG. 1E). In both cases, phage showed little affinity fornormal or hyperplastic skin (FIG. 1E).

To further characterize homing selectivity, dysplasia-homing phagebearing CSRPRRSEC (SEQ ID NO: 3) were intravenously injected into 4 to 6month-old dysplasia-bearing mice. In parallel, 9 to 12 month-oldtumor-bearing mice were infused with the tumor-homing phage CGTKRKC (SEQID NO: 11), CGKRK (SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7). As shown inFIG. 2, phage co-localized in each case with CD31-positive endothelialcells in the expected target tissue. In particular, CSRPRRSEC (SEQ IDNO: 3)-phage accumulated in dysplastic skin (FIG. 2A) of mice bearingdysplasias but no tumors, and CGTKRKC (SEQ ID NO: 11) phage weredetected to a lesser extent in dysplastic skin of tumor-bearing mice(FIG. 2B). In addition, CGKRK (SEQ ID NO: 6) phage accumulated in tumortissue (FIG. 2C), and CDTRL (SEQ ID NO: 7) phage were localized tolarge, dilated vessels throughout dysplastic and hyperplastic skin (FIG.2D) as well as in tumors (FIG. 2E).

Synthetic peptides having homing sequences were also analyzed outside ofthe context of phage particles. Both younger dysplasia-bearing and oldertumor-bearing K14-HPV16 mice were injected with fluorescein-labeledpeptides. As shown in FIG. 3, fluorescein-labeled peptides co-localizedwith the cell-surface endothelial marker Meca-32 in target neoplastictissue after intravenous injection, and were not detected in tissueswhere the corresponding phage did not home. Specifically,fluorescein-labeled CSRPRRSEC (SEQ ID NO: 3) co-localized with Meca-32in dysplastic skin vasculature from both non-tumor-bearing (FIG. 3A) andtumor-bearing mice (FIG. 3D, inset). As is evident in FIG. 3D, peptideSEQ ID NO: 3 was not detected within squamous tumors, confirming theselectivity of this peptide for pre-malignant dysplastic vasculature. Incontrast, fluorescein-labeled CGKRK (SEQ ID NO: 6) and CDTRL (SEQ ID NO:7) peptides were not detected in the dysplastic skin of youngernon-tumor bearing mice (FIGS. 3B and 3C) but were primarily detected intumor vasculature (FIGS. 3E and F), and at lower levels in dysplasticskin of these tumor-bearing mice. Together with the immunolocalizationanalyses of phage homing, the peptide localization data indicate thatCGKRK (SEQ ID NO: 6) and CDTRL (SEQ ID NO: 7) home specifically to bloodvessels in squamous cell carcinomas and in the dysplastic foci oftumor-bearing mice, but not to vasculature of earlier stage dysplasiasin non-tumor bearing mice.

As further disclosed herein in Example IV, homing selectivity ofK14-HPV16 squamous cell cancer-homing peptides, CGKRK (SEQ ID NO: 6) andCDTRL (SEQ ID NO: 7), was analyzed for the ability of these peptides tohome to endothelium in tumors of different tissue origins and localizedto different anatomical locations. In particular, three subcutaneouslyimplanted tumors and two tumors produced in transgenic animal modelswere examined for accumulation of fluorescein-labeled peptides followingintravenous injection. As shown in FIG. 4, different homingspecificities were observed for each peptide in the various tumormicroenvironments. In particular, in FIGS. 4A and F, neither peptideCGKRK (SEQ ID NO: 6) nor CDTRL (SEQ ID NO: 7) homed to angiogenic islets(dysplasias) or tumors in the RIP-Tag transgenic mouse model ofpancreatic islet cell carcinoma (Hanahan, Nature 315:115-122 (1985)),indicating that the binding moieties for these peptides are not presentin normal, dysplastic, or pancreatic tumor vasculature. Both CGKRK (SEQID NO: 6) and CDTRL (SEQ ID NO: 7) did home to breast carcinomas in theMMTV-PyMT transgenic mouse model (Guy et al., Mol. Cell. Biol.12:954-961 (1992)) as shown in FIGS. 4B and G, and also bound a range ofcultured tumor cells in addition to homing to tumor endothelial cells invivo.

The two peptides that homed to vasculature of malignant skin showeddifferent specificity when assayed for the ability to home to threetypes of subcutaneously grown transplanted tumors. Fluorescein-CGKRK(SEQ ID NO: 6) homed to cells in each of the three transplant tumors(FIGS. 4C-E), which arose from PDSC5, a K14-HPV16 tumor-derived cellline (FIG. 4C); the MDA-MB-435 human breast cancer line (FIG. 4D; Priceet al., Cancer Res. 50:717-721 (1990)); or the C8161 human melanoma line(FIG. 4E; Bregman and Meyskens, Int. J. Cancer 37:101-107 (1986)). Incontrast, the CDTRL (SEQ ID NO: 7) peptide accumulated only in themelanoma xenografts (FIG. 4J) and in the skin overlying the melanomaxenograft tumor (FIG. 4J inset). Furthermore, fluorescein-CGKRK (SEQ IDNO: 6) localized in the cytoplasm and nuclei of vascular cellsidentified as endothelial cells by their morphology and byimmunostaining for CD31 and Meca-32 (see FIG. 4D). These resultsindicate that cell type or oncogenic stimulus imparts differentqualities onto vasculature and the tumor microenvironment, as revealedby differential selective homing patterns.

The discoveries disclosed herein relate to homing peptides and otherhoming molecules that target different stages of tumor developmentincluding, but not limited to, squamous cell carcinogenesis and otherdermatological malignancies. Such molecules can distinguish betweentemporal changes that occur during multi-stage tumorigenesis and provideimproved diagnostic tools, including ones affording early detection ofpre-malignant lesions and asymptomatic early stage carcinomas. Suchhoming molecules also can be useful for specifically targetingtherapeutics to vasculature of early-stage, pre-malignant lesions orvasculature of a later-stage dysplasia or tumor.

Based on the discoveries discussed above, the present invention providesan isolated peptide or peptidomimetic which has a length of less than 20residues and includes the amino acid sequence SRPRR (SEQ ID NO: 1) or apeptidomimetic thereof. Such a peptide or peptidomimetic can be cyclicor otherwise conformationally constrained. In particular embodiments,the peptide or peptidomimetic includes the amino acid sequence CSRPRRSEC(SEQ ID NO: 3), CSRPRRSVC (SEQ ID NO: 4) or CSRPRRSWC (SEQ ID NO: 5) ora peptidomimetic thereof. An isolated peptide or peptidomimetic of theinvention, such as a conformationally constrained peptide orpeptidomimetic can have, without limitation, a length of less than 15residues or a length of less than 10 residues. A peptide orpeptidomimetic of the invention can selectively home to vasculature ofpre-malignant dysplastic skin.

The present invention further provides an isolated peptide orpeptidomimetic which has a length of less than 90 residues and whichincludes the amino acid sequence CXSRPRRZC (SEQ ID NO: 2) or apeptidomimetic thereof, where X=0 to 20 independently selected residuesand Z=0 to 20 independently selected residues. Such an isolated peptideor peptidomimetic can be cyclic or otherwise conformationallyconstrained and can include, without limitation, the amino acid sequenceCSRPRRSEC (SEQ ID NO: 3), CSRPRRSVC (SEQ ID NO: 4) or CSRPRRSWC (SEQ IDNO: 5) or a peptidomimetic thereof. An isolated peptide orpeptidomimetic of the invention can have, without limitation, a lengthof less than 60 residues, a length of less than 40 residues, or a lengthof less than 30 residues. Any of such peptides or peptidomimeticsincluding the amino acid sequence CXSRPRRZC (SEQ ID NO: 2) or apeptidomimetic thereof can selectively home to vasculature ofpre-malignant dysplastic skin.

Further provided by the present invention is an isolated peptide orpeptidomimetic which has a length of less than 20 residues and whichincludes the amino acid sequence CGKRK (SEQ ID NO: 6) or apeptidomimetic thereof. An isolated peptide or peptidomimetic of theinvention, such as a conformationally constrained peptide orpeptidomimetic can have, without limitation, a length of less than 15residues or a length of less than 10 residues. In one embodiment, theisolated peptide or peptidomimetic is cyclic. In another embodiment, theisolated peptide or peptidomimetic is conformationally constrained. Anyof such peptides or peptidomimetics including the amino acid sequenceCGKRK (SEQ ID NO: 6) or a peptidomimetic thereof can selectively home tovasculature of malignant skin.

Additionally provided herein is an isolated peptide or peptidomimeticwhich has a length of less than 20 residues and which includes the aminoacid sequence CDTRL (SEQ ID NO: 7) or a peptidomimetic thereof. Anisolated peptide or peptidomimetic of the invention, such as aconformationally constrained peptide or peptidomimetic can have, forexample, a length of less than 15 residues or a length of less than 10residues. In particular embodiments, the isolated peptide orpeptidomimetic is cyclic or conformationally constrained. A peptide orpeptidomimetic of the invention including the amino acid sequence CDTRL(SEQ ID NO: 7) or a peptidomimetic thereof can selectively home tovasculature of malignant skin.

The peptides and peptidomimetics of the invention are provided inisolated form. As used herein in reference to a peptide orpeptidomimetic of the invention, the term “isolated” means a peptide orpeptidomimetic that is in a form that is relatively free from materialsuch as contaminating polypeptides, lipids, nucleic acids and othercellular material that normally is associated with the peptide orpeptidomimetic in a cell or that is associated with the peptide orpeptidomimetic in a library or in a crude preparation.

Further provided by the present invention is a conjugate that contains atherapeutic moiety linked to a homing peptide or peptidomimetic whichselectively homes to vasculature of pre-malignant dysplastic skin andwhich includes the amino acid sequence SRPRR (SEQ ID NO: 1) or aconservative variant or peptidomimetic thereof.

Also provided herein is a conjugate containing a therapeutic moietylinked to a homing peptide or peptidomimetic which selectively homes tovasculature of malignant skin and which includes the amino acid sequenceCGKRK (SEQ ID NO: 6) or the amino acid sequence CDTRL (SEQ ID NO: 7), ora conservative variant or peptidomimetic of one of these sequences.

The conjugates and methods of the invention disclosed herein below relyon homing molecules. As used herein, the term “molecule” is used broadlyto mean a polymeric or non-polymeric organic chemical such as a smallmolecule drug; a nucleic acid molecule such as an RNA, a cDNA or otherDNA, or an oligonucleotide; a peptide or peptidomimetic; or a proteinsuch as an antibody or a growth factor receptor or a fragment thereofsuch as an Fv, Fd, or Fab fragment of an antibody containing theantigen-binding domain.

The phrase “homing molecule that selectively homes to vasculature ofpre-malignant dysplastic skin,” as used herein, means any molecule thatpreferentially localizes in vivo to vasculature of pre-malignantdysplastic skin as compared to vasculature of malignant skin andvasculature of normal skin. Similarly, the phrase “homing peptide orpeptidomimetic that selectively homes to vasculature of pre-malignantdysplastic skin” means a peptide or peptidomimetic that preferentiallylocalizes in vivo to vasculature of pre-malignant dysplastic skin ascompared to vasculature of malignant skin and vasculature of normalskin. It is understood that a homing molecule that selectively homes tovasculature of pre-malignant dysplastic skin can home to the supportingvasculature of a variety of dysplastic lesions in addition to dysplasticskin, or can exhibit preferential homing to vasculature of pre-malignantdysplastic lesions in a subset of tissue types including dysplasticskin, or can exhibit significant homing exclusively to vasculature ofpre-malignant dysplastic skin.

Selective homing of a homing molecule that selectively homes tovasculature of pre-malignant dysplastic skin generally is characterizedby at least a two-fold greater localization within vasculature ofpre-malignant dysplastic skin as compared to vasculature of malignantskin and normal skin vasculature. Such a homing molecule can becharacterized, for example, by 5-fold, 10-fold, 20-fold or more greaterlocalization within vasculature of pre-malignant dysplastic skin ascompared to vasculature of malignant skin and normal skin vasculature.As discussed above, it is understood that a homing molecule thatselectively homes to vasculature of pre-malignant dysplastic skin canhome, in part, to vasculature of one or more other dysplastic tissues.

The phrase “homing molecule that selectively homes to vasculature ofmalignant skin,” as used herein, means any molecule that preferentiallylocalizes in vivo to vasculature of malignant skin or later-stagedysplasias as compared to vasculature of early-stage pre-malignantdysplasias and vasculature of normal skin. Similarly, the phrase “homingpeptide or peptidomimetic that selectively homes to vasculature ofmalignant skin” means a peptide or peptidomimetic that preferentiallylocalizes in vivo to vasculature of malignant skin or later-stagedysplasias as compared to vasculature of early-stage pre-malignantdysplasias and vasculature of normal skin. One skilled in the artunderstands that a homing molecule that selectively homes to vasculatureof malignant skin can home to the supporting vasculature of a variety ofdifferent malignancies in addition to malignant skin, or can exhibitpreferential homing to vasculature of a subset of malignancies includingmalignant skin, or can exhibit signficant homing exclusively tovasculature of malignant skin.

Selective homing of a homing molecule that selectively homes tovasculature of malignant skin generally is characterized by at least atwo-fold greater localization within vasculature of malignant skin orlater-stage dysplasias as compared to vasculature of early-stagepre-malignant dysplastic skin and normal skin. Such a homing moleculecan be characterized, for example, by 5-fold, 10-fold, 20-fold or moregreater localization within vasculature of malignant skin or later-stagedysplasias as compared to vasculature of early-stage pre-malignantdysplastic skin and normal skin. As discussed above, it is understoodthat a homing molecule that selectively homes to vasculature ofmalignant skin can additionally localize to vasculature or tumor cellsof one or more other malignant tissues in addition to selectively homingto malignant skin.

The present invention also provides a conjugate containing a homingpeptide or peptidomimetic that includes an amino acid sequence which isa conservative variant, for example, of the amino acid sequence SRPRR(SEQ ID NO: 1). As used herein, a “conservative variant” is an aminoacid sequence in which a first amino acid is replaced by a second aminoacid or amino acid analog having at least one similar biochemicalproperty, which can be, for example, similar size, charge,hydrophobicity or hydrogen-bonding capacity. For example, a firsthydrophobic amino acid can be conservatively substituted with a second(non-identical) hydrophobic amino acid such as alanine, valine, leucine,or isoleucine, or an analog thereof. Similarly, a first basic amino acidcan be conservatively substituted with a second basic amino acid such asarginine or lysine, or an analog thereof. In the same way, a firstacidic amino acid can be conservatively substituted with a second acidicamino acid such as aspartic acid or glutamic acid, or an analog thereof,or an aromatic amino acid such as phenylalanine can be conservativelysubstituted with a second aromatic amino acid or amino acid analog, forexample, tyrosine.

Further provided herein is a multivalent conjugate containing a moietylinked to at least two homing peptides or peptidomimetics which eachselectively home to vasculature of pre-malignant dysplastic skin andwhich include the amino acid sequence SRPRR (SEQ ID NO: 1) or aconservative variant or peptidomimetic thereof. Such a multivalentconjugate can include, without limitation, at least 10 homing peptidesor peptidomimetics or at least 100 homing peptides or peptidomimetics.In a multivalent conjugate of the invention, the homing peptides orpeptidomimetics can optionally be cyclic or otherwise conformationallyconstrained, and can be linked to any of a variety of moieties such as aphage moiety.

In one embodiment, the invention provides a multivalent conjugatecontaining a moiety linked to at least two homing peptides orpeptidomimetics which each selectively home to vasculature ofpre-malignant dysplastic skin and which each independently include theamino acid sequence CXSRPRRZC (SEQ ID NO: 2) or a conservative variantor peptidomimetic thereof, where X=0 to 20 independently selectedresidues and where Z=0 to 20 independently selected residues. In furtherembodiments, such a multivalent conjugate includes at least ten homingpeptides or peptidomimetics that each selectively home to vasculature ofpre-malignant dysplastic skin, or at least 100 homing peptides orpeptidomimetics that each selectively home to vasculature ofpre-malignant dysplastic skin. Moieties useful in a multivalentconjugate of the invention include but are not limited to phagemoieties. In further embodiments, the invention provides a multivalentconjugate containing a moiety linked to at least two, at least ten, orat least 100, homing peptides or peptidomimetics which each selectivelyhome to vasculature of pre-malignant dysplastic skin and which eachindependently include the amino acid sequence CSRPRRSEC (SEQ ID NO: 3)or a conservative variant or peptidomimetic thereof. In otherembodiments, the invention provides a multivalent conjugate containing amoiety linked to at least two, at least ten, or at least 100, homingpeptides or peptidomimetics which each selectively home to vasculatureof pre-malignant dysplastic skin and which each independently includethe amino acid sequence CSRPRRSVC (SEQ ID NO: 4) or a conservativevariant or peptidomimetic thereof. In still further embodiments, theinvention provides a multivalent conjugate containing a moiety linked toat least two, at least ten, or at least 100, homing peptides orpeptidomimetics which each selectively home to vasculature ofpre-malignant dysplastic skin and which each independently include theamino acid sequence CSRPRRSWC (SEQ ID NO: 5) or a conservative variantor peptidomimetic thereof.

Also provided by the invention is a multivalent conjugate containing amoiety linked to at least two homing peptides or peptidomimetics whicheach selectively home to vasculature of malignant skin and which includethe amino acid sequence CGKRK (SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7) ora conservative variant or peptidomimetic thereof. Such a multivalentconjugate can include, without limitation, at least 10 homing peptidesor peptidomimetics or at least 100 homing peptides or peptidomimetics.In a multivalent conjugate of the invention, the homing peptides orpeptidomimetics can optionally be cyclic or otherwise conformationallyconstrained, and can be linked to any of a variety of moieties such as aphage moiety.

A multivalent conjugate of the invention containing multiple homingmolecules can include, for example, two or more, three or more, five ormore, ten or more, twenty or more, thirty or more, forty or more, fiftyor more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or moreor 1000 or more homing molecules. In one embodiment, the homingmolecules have an identical amino acid sequence. In another embodiment,the multivalent conjugate includes homing molecules having non-identicalamino acid sequences. Moieties useful in a multivalent conjugate of theinvention include, without limitation, phage, retroviruses,adenoviruses, adeno-associated viruses and other viruses, cells,liposomes, polymeric matrices, non-polymeric matrices or particles suchas gold particles, microdevices and nanodevices, and nano-scalesemiconductor materials.

Liposomes consisting, without limitation, of phospholipids or otherlipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are readily made to be incorporated into a conjugate ofthe invention (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, BocaRaton, Fla. (1984)). It is understood that the liposome or otherpolymeric matrix additionally can include one or more other componentsif desired, such as, without limitation, one or any combination oftherapeutic agents, anti-angiogenic agents or cytotoxic agents.

As disclosed herein, peptide CSRPRRSEC (SEQ ID NO: 3) recognizes atarget “receptor” which is expressed in vasculature of pre-malignantskin dysplasias but which is essentially absent or inaccessible forbinding via the circulation in vasculature of normal skin andvasculature of squamous cell carcinomas. The cell surface and cell-typeselective expression of the target receptor form the basis for theselective homing activity of peptide SEQ ID NO: 3, 4 and 5 and relatedpeptides, peptidomimetics and other molecules. Based on this discovery,it is also clear that molecules structurally unrelated to SEQ ID NO: 3,4 or 5 or the generic sequences SEQ ID NOS: 1 and 2 but which bind thesame receptor also have the same characteristic of selectively homing tovasculature of pre-malignant dysplastic skin and other pre-malignantdysplastic tissues. Such molecules can be identified by the ability tospecifically bind to, or to compete with SEQ ID NO: 3, 4, 5 or a relatedpeptide such as a SRPRR (SEQ ID NO: 1)-containing peptide for specificbinding to, cells expressing a cognate receptor for SEQ ID NO: 3 such aspre-malignant dysplastic skin cells from K14-HPV16 mice at 4-6 months ofage as described further below. Selective homing to vasculature ofpre-malignant dysplastic skin readily can be confirmed using in vivopanning as disclosed herein in Example I (see, also, U.S. Pat. No.5,622,699).

A homing molecule of the invention specifically binds the indicatedcognate receptor. As used herein, the term “specifically binds” or“specifically binding” means binding that is measurably different from anon-specific interaction. Specific binding can be measured, for example,by determining binding of a molecule compared to binding of a controlmolecule, which generally is a molecule of similar structure that doesnot have binding activity. In this case, specific binding is indicatedif the molecule has measurably higher affinity for cells expressing thecognate receptor, for example, than for cells that do not express thecognate receptor. As a non-limiting example, peptides SEQ ID NOS: 6 and7 bind a range of cultured tumor cells in vivo, including, for example,HL-60 human leukemia cells, MDA-MB-435 human breast carcinomas and PDSC5squamous cell carcinomas. Specificity of binding can be determined, forexample, by competitive inhibition of the binding of a known bindingmolecule such as SEQ ID NO: 3 to identify molecules that selectivelyhome to vasculature of pre-malignant dysplastic skin, or by competitiveinhibition of the binding of a known binding molecule such as SEQ ID NO:6 or 7 to identify molecules that selectively home to vasculature ofmalignant skin.

The term “specifically binding,” as used herein, includes both low andhigh affinity specific binding. Specific binding can be exhibited, forexample, by a low affinity homing molecule having a Kd of at least about10⁻⁴ M. For example, if the cognate receptor has more than one bindingsite, a homing molecule having low affinity can be useful for targeting,for example, vasculature of pre-malignant dysplastic skin or vasculatureof malignant skin. Specific binding also can be exhibited by a highaffinity homing molecule, for example, a homing molecule having a Kd ofat least about 10⁻⁵ M. Such a molecule can have, for example, a Kd of atleast about 10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, atleast about 10⁻⁹ M, at least about 10⁻¹⁰M, or can have a Kd of at leastabout 10⁻¹ M or 10⁻¹² M or greater. Both low and high affinity homingmolecules are useful and are encompassed by the invention. Low affinityhoming molecules are useful in targeting, for example, multivalentconjugates such as viruses and other particles. High affinity homingmolecules are useful in targeting, for example, multivalent andunivalent conjugates.

Thus, the invention further provides a conjugate which contains atherapeutic moiety linked to a homing molecule which selectively homesto vasculature of pre-malignant dysplastic skin and which specificallybinds a cognate receptor for CSRPRRSEC (SEQ ID NO: 3). In oneembodiment, such a conjugate contains a homing molecule which is not anantibody or antigen-binding fragment thereof. In another embodiment, thehoming molecule is a kallikrein 9-binding molecule. In furtherembodiments, the peptide or peptidomimetic portion of the conjugate hasa length of at most 200 residues, or a length of at most 50 residues.

Also provided by the invention is a conjugate which contains adetectable moiety linked to a homing molecule which selectively homes tovasculature of pre-malignant dysplastic skin and which specificallybinds a cognate receptor for CSRPRRSEC (SEQ ID NO: 3). Such a conjugatecan contain, for example, a homing molecule which is not an antibody orantigen-binding fragment thereof. In another embodiment, the homingmolecule is a kallikrein 9-binding molecule. In further embodiments, thepeptide or peptidomimetic portion of the conjugate has a length of atmost 200 residues, or a length of at most 50 residues. A variety ofdetectable moieties are useful in a conjugate of the inventionincluding, but not limited to, radionuclides and fluorescent labels.

A homing molecule which specifically binds a cognate receptor forCSRPRRSEC (SEQ ID NO: 3) can be, without limitation, a homing peptide orpeptidomimetic. In one embodiment, a conjugate of the invention containsa homing peptide or peptidomimetic which selectively homes tovasculature of pre-malignant dysplastic skin and which includes theamino acid sequence SRPRR (SEQ ID NO: 1) or a conservative variant orpeptidomimetic of this sequence. Such a homing peptide or peptidomimeticcan include, for example, the amino acid sequence SEQ ID NO: 1, or apeptidomimetic thereof. In another embodiment, a conjugate of theinvention contains a homing peptide or peptidomimetic which selectivelyhomes to vasculature of pre-malignant dysplastic skin and which includesthe amino acid sequence SEQ ID NO: 2, where X=0 to 20 independentlyselected residues and where Z=0 to 20 independently selected residues,or a conservative variant or peptidomimetic thereof. Such a homingpeptide or peptidomimetic can include, for example, the amino acidsequence SEQ ID NO: 2, where X=0 to 20 independently selected residuesand where Z=0 to 20 independently selected residues, or a peptidomimeticof this sequence.

There further is provided herein a method of directing a moiety tovasculature of a pre-malignant dysplastic tissue in a subject byadministering to the subject a conjugate that contains a moiety linkedto a homing molecule which selectively homes to vasculature ofpre-malignant dysplastic skin and which specifically binds a cognatereceptor for CSRPRRSEC (SEQ ID NO: 3), thereby directing the moiety tovasculature of the pre-malignant dysplastic tissue. In one embodiment,the invention provides a method of directing a moiety to vasculature ofa pre-malignant dysplastic tissue using a homing molecule other than anantibody or antigen-binding fragment thereof.

The present invention further provides a method of imaging vasculatureof a pre-malignant dysplastic tissue by administering to the subject aconjugate containing a detectable moiety linked to a homing moleculewhich selectively homes to vasculature of pre-malignant dysplastic skinand which specifically binds a cognate receptor for CSRPRRSEC (SEQ IDNO: 3) and detecting the conjugate, thereby imaging vasculature of thepre-malignant dysplastic tissue. In one embodiment, the inventionprovides a method of imaging vasculature of a pre-malignant dysplastictissue using a homing molecule other than an antibody or antigen-bindingfragment thereof.

Further provided herein is a method of reducing the risk of progressionto a malignancy in a subject by administering to the subject a conjugatecontaining a therapeutic moiety linked to a homing molecule whichselectively homes to vasculature of pre-malignant dysplastic skin andwhich specifically binds a cognate receptor for CSRPRRSEC (SEQ ID NO:3), thereby diminishing vasculature of a pre-malignant dysplastic tissueand reducing the risk of progression to the malignancy. In oneembodiment, such a method of reducing the risk of progression to amalignancy is practiced with a homing molecule other than an antibody orantigen-binding fragment thereof.

Moieties useful in the methods of the invention are discussed furtherbelow and include, without limitation, therapeutic and detectablemoieties. Therapeutic moieties which can be directed to vasculature of apre-malignant dysplastic lesion include, but are not limited to,anti-angiogenic agents such as those effective against pre-malignantvasculature, and cytotoxic agents. Moieties which can be directed tovasculature of a pre-malignant dysplastic lesion further encompass,without limitation, detectable moieties such as fluorescent labels andradionuclides, including but not limited to, indium-111, technetium-99,carbon-11, and carbon-13.

In particular embodiments, the conjugates and methods of the inventionare practiced with a homing molecule which selectively homes tovasculature of pre-malignant dysplastic skin and which specificallybinds a cognate receptor for CSRPRRSEC (SEQ ID NO: 3), where the homingmolecule does not include any of the amino acid sequences shown in Table1 below. In further embodiments, the conjugates and methods of theinvention are practiced with a homing molecule which selectively homesto vasculature of pre-malignant dysplastic skin and which specificallybinds a cognate receptor for CSRPRRSEC (SEQ ID NO: 3), where the homingmolecule does not include any of the amino acid sequences shown in Table1, or conservative variants thereof. TABLE I CYADCEGTCGMVC (18)CWNICPGGCRALC (19) GPGCEEECQPAC (20) CKGTCVLGCSEEC (21) CSTLCGLRCMGTC(22) CMPRCGVNCKWAC (23) CVGACDLKCTGGC (24) CVALCREACGEGC (25)CSSGCSKNCLEMC (26) CGRPCRGGCAASC (27) CQGGCGVSCPIFC (28) CAVRCDGSCVPEC(29) CGFGCSGSCQMQC (30) CRVVCADGCRFIC (31) CTMGCTAGCAFAC (32)CEGKCGLTCECTC (33) CNQGCSGSCDVMC (34) CASGCSESCYVGC (35) CGGGCQWGCAGEC(36) CSVRCKSVCIGLC (37) CPSNCVALCTSGC (38) CVEGCSSGCGPGC (39)CRVVCADGCRLIC (40) CSTLCGLRCMGTC (41) CFTFCEYHCQLTC (42)Parentheses contain SEQ ID NO:.

Homing molecules which selectively home to vasculature of pre-malignantdysplastic skin and which selectively bind a cognate receptor for SEQ IDNO: 3 include, but are not limited to, kallikrein 9 analogs such assmall molecule, peptide and peptidomimetic analogs. Kallikrein 9 (KLK9or KLK-L3) is a member of the kallikrein gene family, a subfamily ofserine proteases with a conserved catalytic triad (histidine, asparticacid and serine). At least 14 human kallikrein-like genes have beenidentified, including the prognostic marker prostate-specific antigen(PSA), and all colocalize to the same region of chromosome 13(q13.3-13.4). Members of the kallikrein gene family generally have fivecoding exons and exhibit 30-80% sequence homology at the DNA and aminoacid levels, with kallikrein 9 exhibiting 38% and 33% amino acididentity with the KLK-L2 and KLK-L1 proteins, respectively. Many membersof the kallikrein gene family, including kallikrein 9, are upregulatedby steroid hormones and further may be downregulated in breast or othercancers or have anti-angiogenic activity (Diamandis et al., TrendsEndocrin. Metab. 11: 54-60 (2000)).

Kallikrein 9 is mainly expressed in skin, thymus, trachea, cerebellumand spinal cord as well as brain, salivary gland, mammary gland, ovaryand prostate. (Yousef et al., Genomics 65:184-194 (2000); Yousef et al.,Anticancer Res. 19:2843-2852 (1999)). Lower levels of kallikrein 9expression are observed in fetal brain, stomach, lung, thyroid,placenta, liver, small intestine and bone marrow (Yousef et al., supra,2000). As indicated above, kallikrein 9 is hormonally regulated, forexample, by the steroids estrogen and progestin in ovarian and breastcancer cell lines. Furthermore, in ovarian tumors, kallikrein 9expression is an independent favorable prognostic marker; patients withKLK9-positive tumors have substantially increased progression-freesurvival as well as substantially increased overall survival (Yousefetal., Cancer Res. 61: 7811-7817 (2001)).

As disclosed herein, the Cys220 to Arg225 portion of human kallikrein 9(C₂₂₀SRPRR₂₂₅ (SEQ ID NO: 17); Yousef and Diamandis, Genomics 65:184-194(2000)) is homologous to the homing peptide CSRPRRSEC (SEQ ID NO: 3).The Cys200 to Arg225 sequence is also conserved in the mouse homolog ofkallikrein 9 (RIKEN clone 1200016C12; Kawai et al., Nature 409:685-690(2001)) and is present in homing peptides CSRPRRSVC (SEQ ID NO: 4) andCSRPRRSWC (SEQ ID NO: 5), which, like CSRPRRSEC (SEQ ID NO: 3),selectively home to vasculature of pre-malignant dysplastic skin.Structural studies have shown that the Cys200 to Arg225 sequence ofhuman kallikrein 9 forms a loop that defines substrate specificities inthe kallikreins by contributing to a portion of the entrance to theactive site; this loop is highly variable among kallikreins and othertrypsin-family members (Gomis-Ruth et al., J. Biol. Chem.277:27273-27281 (2002)). In kallikrein 9 and other kallikreins, Cys200forms a disulfide bond with Cys190, the cysteine next to the active sitenucleophile Ser195; the loop is closed with a kink introduced byinvariant Pro226. Similarly, a disulfide bond is present in CSRPRRSEC(SEQ ID NO: 3), CSRPRRSVC (SEQ ID NO: 4) and CSRPRRSWC (SEQ ID NO: 5),conformationally constraining these homing peptides, which can act asanalogs of kallikrein 9.

As used herein, the term “kallikrein 9 analog” means a molecule thatspecifically binds to a naturally occurring kallikrein 9-bindingmolecule which is expressed or localized at higher levels on angiogenicendothelia of pre-malignant dysplastic skin lesions than on angiogenicendothelia of squamous carcinomas. Such a kallikrein 9 analog can be,without limitation, a small molecule, peptide, peptidomimetic or proteinthat specifically binds to a naturally occurring kallikrein 9-bindingmolecule, which is a kallikrein 9 substrate or inhibitor. In oneembodiment, the kallikrein 9 analog specifically binds to a substratewhich is a precurosr to an anti-angiogenic molecule such as prostateserum antigen (PSA). In another embodiment, the kallikrein 9 analogspecifically binds to a substrate which is a pro-angiogenic factorinactivated upon cleavage by kallikrein 9. Kallikrein 9 analogs include,but are not limited to, peptides and peptidomimetics such as SEQ ID NO:1; SEQ ID NO: 2, where X=0 to 20 independently selected residues andwhere Z=0 to 20 independently selected residues; SEQ ID NO: 3; SEQ IDNO: 4; and SEQ ID NO: 5, and conservative variants and peptidomimeticsof any of these sequences.

As further disclosed herein, peptides CGKRK (SEQ ID NO: 6) and CDTRL(SEQ ID NO: 7) each recognize a target “receptor” which is expressed invasculature of malignant skin and breast carcinomas but which isessentially absent or inaccessible for binding via the circulation innormal skin and other normal tissues and present at reduced or variablelevels in early stage dysplasias. The cell surface and cell-typeselective expression of the target receptor form the basis for theselective homing activity of peptides SEQ ID NOS: 6 and 7 and relatedpeptides, peptidomimetics and other molecules. From the above, it isclear to one skilled in the art that molecules structurally unrelated toSEQ ID NO: 6 or 7 but which bind one of the same cognate receptors alsohave the same characteristic of selectively homing to vasculature ofmalignant tissues such as vasculature of malignant skin. Such moleculescan be identified by the ability to specifically bind to, or to competewith SEQ ID NO: 6 or 7 for specific binding to, cells expressing thecognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7, such as MDA-MB-435human breast cancer cells, C8161 human melanoma cells, HL-60 humanleukemia cells and PDSC5 squamous cell carcinomas. Selective homing tovasculature of malignant tissues readily can be confirmed using in vivopanning (see Example II and U.S. Pat. No. 5,622,699).

The invention further provides a conjugate which contains a therapeuticmoiety linked to a homing molecule which selectively homes tovasculature of malignant skin and which specifically binds a cognatereceptor for CGKRK (SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7). In oneembodiment, such a conjugate contains a homing molecule which is not anantibody or antigen-binding fragment thereof. In further embodiments,the peptide or peptidomimetic portion of the conjugate has a length ofat most 200 residues, or a length of at most 50 residues.

Also provided herein is a conjugate which contains a detectable moietylinked to a homing molecule which selectively homes to vasculature ofmalignant skin and which specifically binds a cognate receptor for CGKRK(SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7). In one embodiment, such aconjugate contains a homing molecule which is not an antibody orantigen-binding fragment thereof. In further embodiments, the peptide orpeptidomimetic portion of the conjugate has a length of at most 200residues, or a length of at most 50 residues. Detectable moieties usefulin a conjugate of the invention include, without limitation,radionuclides and fluorescent labels.

A homing molecule which specifically binds a cognate receptor for CGKRK(SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7) can be, without limitation, ahoming peptide or peptidomimetic. In one embodiment, a conjugate of theinvention contains a homing peptide or peptidomimetic which selectivelyhomes to vasculature of malignant skin and which is cyclic or otherwiseconformationally constrained. In another embodiment, a conjugate of theinvention contains a homing peptide or peptidomimetic which selectivelyhomes to vasculature of malignant skin and which includes the amino acidsequence CGKRK (SEQ ID NO: 6) or a conservative variant orpeptidomimetic of this sequence. Such a homing peptide or peptidomimeticcan include, for example, the amino acid sequence SEQ ID NO: 6, or apeptidomimetic thereof. In another embodiment, a conjugate of theinvention contains a homing peptide or peptidomimetic which selectivelyhomes to vasculature of malignant skin and which includes the amino acidsequence CDTRL (SEQ ID NO: 7), or a conservative variant orpeptidomimetic thereof. Such a homing peptide or peptidomimetic cancontain, for example, the amino acid sequence SEQ ID NO: 7 or apeptidomimetic thereof.

Also provided herein is a method of directing a moiety to vasculature ofa malignant tissue in a subject by administering to the subject aconjugate containing a moiety linked to a homing molecule whichselectively homes to vasculature of malignant skin and whichspecifically binds a cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7,thereby directing the moiety to vasculature of the malignant tissue. Inone embodiment, a method of the invention for directing a moiety tovasculature of a malignant tissue in a subject is practiced with ahoming molecule other than an antibody or antigen-binding fragmentthereof.

Also provided herein is a method of imaging vasculature of a malignanttissue by administering to the subject a conjugate containing adetectable moiety linked to a homing molecule which selectively homes tovasculature of malignant skin and which specifically binds a cognatereceptor for SEQ ID NO: 6 or SEQ ID NO: 7; and detecting the conjugate,thereby imaging vasculature of the malignant tissue. The inventionprovides, in one embodiment, a method of imaging vasculature of amalignant tissue using a homing molecule other than an antibody orantigen-binding fragment thereof.

In addition, there is provided herein a method of treating a cancer in asubject by administering to the subject a conjugate containing atherapeutic moiety linked to a homing molecule which selectively homesto vasculature of malignant skin and which specifically binds a cognatereceptor for SEQ ID NO: 6 or SEQ ID NO: 7, thereby directing thetherapeutic moiety to vasculature of the cancer and treating the cancer.In one embodiment, the invention provides a method of treating cancer ina subject using a homing molecule other than an antibody orantigen-binding fragment thereof.

The present invention further provides a method of staging tumorprogression in a subject having or suspected of having a pre-malignantlesion or tumor by administering to the subject a conjugate containing adetectable moiety linked to (i) a homing molecule which selectivelyhomes to vasculature of pre-malignant dysplastic skin and whichspecifically binds a cognate receptor for CSRPRRSEC (SEQ ID NO: 3), or(ii) a homing molecule which selectively homes to vasculature ofmalignant skin and which specifically binds a cognate receptor for CGKRK(SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7); and detecting the conjugate,where detection of the conjugate containing a homing molecule whichselectively homes to vasculature of pre-malignant dysplastic skinindicates a pre-malignant stage of tumor progression in the subject andwhere detection of the conjugate containing a homing molecule whichselectively homes to vasculature of malignant skin indicates a malignantstage of tumor progression in the subject. In one embodiment, such amethod of staging tumor progression is practiced with a homing moleculeother than an antibody or antigen-binding fragment thereof.

A variety of moieties can be linked to a homing molecule thatselectively homes to vasculature of malignant skin in a method of theinvention. Such moieties, which are discussed further below include,without limitation, therapeutic and detectable moieties. Therapeuticmoieties which can be directed to vasculature of a malignant tissueinclude, but are not limited to, anti-angiogenic agents such as thoseeffective against tumor vasculature, and cytotoxic agents. Moietieswhich can be directed to vasculature of a malignant tissue and which canbe useful in staging tumor progression further encompass, withoutlimitation, detectable moieties such as fluorescent labels andradionuclides including, but not limited to, indium-111, technetium-99,carbon-11, and carbon-13.

In particular embodiments, the conjugates and methods of the inventionare practiced with a homing molecule which selectively homes tovasculature of malignant skin and which specifically binds a cognatereceptor for SEQ ID NO: 6 or SEQ ID NO: 7, where the homing moleculedoes not include any of the amino acid sequences shown in Table 1 above.In further embodiments, the conjugates and methods of the invention arepracticed with a homing molecule which selectively homes to vasculatureof malignant skin and which specifically binds a cognate receptor forSEQ ID NO: 6 or SEQ ID NO: 7, where the homing molecule does not includeany of the amino acid sequences shown in Table 1, or conservativevariants thereof.

As set forth above, in particular embodiments, several conjugates of theinvention include a homing molecule that is not an antibody orantigen-binding fragment thereof. “Antibody” is an art-recognized termthat refers to a peptide or polypeptide containing one or morecomplementarity determining regions (CDRs). See, for example,Borrabaeck, Antibody Engineering 2nd Edition, Oxford University Press,New York (1995).

The invention further provides a multivalent conjugate contain, forexample, a liposome or other polymeric matrix or moiety linked to atleast two homing molecules which each selectively homes to vasculatureof pre-malignant dysplastic skin and which specifically bind a cognatereceptor for SEQ ID NO: 3. Additional multivalent conjugates of theinvention provided herein include a liposome or other polymeric matrixor moiety linked to at least two homing molecules which each selectivelyhomes to vasculature of malignant skin and which specifically bind acognate receptor for SEQ ID NO: 6. Still further multivalent conjugatesprovided herein contain, for example, a liposome or other polymericmatrix or moiety linked to at least two homing molecules which eachselectively homes to vasculature of malignant skin and whichspecifically bind a cognate receptor for SEQ ID NO: 7. If desired, theliposome or other polymeric matrix can be linked to at least ten or atleast 100 of such homing molecules. Any of a variety of moieties can beuseful in such a multivalent conjugate including, but not limited to,those described herein above.

The peptides and peptidomimetics and homing peptides and peptidomimeticsof the invention, including the bifunctional and multivalent peptidesand peptidomimetics disclosed herein below, can have a variety oflengths. A peptide or peptidomimetic of the invention can have, forexample, a relatively short length of less than six, seven, eight, nine,ten, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70 or 80 residues. Apeptide or peptidomimetic of the invention also can be useful in thecontext of a significantly longer sequence as described further below.As used herein, the term “residue” refers to amino acids or analogsthereof. It is understood that a peptide containing, for example, theamino acid sequence SEQ ID NO: 1 includes the specified amino acids as acontiguous sequence not separated by other amino acids.

In other embodiments, the peptide or peptidomimetic portion of theconjugate has a defined length. The peptide or peptidomimetic portion ofthe conjugate can have, for example, a length of at most 10, 20, 30, 40,50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 or 2000residues. It is understood that the term “peptide or peptidomimeticportion of the conjugate” means the total number of residues in thehoming peptide or peptidomimetic and any contiguous protein, peptide orpeptidomimetic, such as a therapeutic protein or pro-apoptotic peptide.

As disclosed herein, a homing peptide or peptidomimetic of the inventioncan maintain homing activity in the context of a significantly longersequence. As a non-limiting example, the 9-mer peptide CSRPRRSRC (SEQ IDNO: 3) maintained the ability to home when fused to a phage coatprotein, confirming that a peptide of the invention can have selectivehoming activity when embedded in a larger protein sequence. Thus, theinvention further provides a chimeric protein containing a peptide orpeptidomimetic of the invention, or a homing peptide or peptidomimeticof the invention, fused to a heterologous protein. In one embodiment,the invention provides a chimeric protein containing a homing peptide orpeptidomimetic which selectively homes to vasculature of pre-malignantdysplastic skin and which specifically binds a cognate receptor forCSRPRRSEC (SEQ ID NO: 3) fused to a heterologous protein. Such aheterologous protein can be, without limitation, a heterologous proteinhaving a therapeutic activity, or an antibody or antigen-bindingfragment thereof. In other embodiments, the invention provides achimeric protein in which a homing peptide or peptidomimetic containingthe amino acid sequence SRPRR (SEQ ID NO: 1), CXSRPRRZC (SEQ ID NO: 2),CSRPRRSEC (SEQ ID NO: 3), CSRPRRSVC (SEQ ID NO: 4) or CSRPRRSWC (SEQ IDNO: 5) or a conservative variant or peptidomimetic of one or thesesequences, is fused to a heterologous protein.

The invention additionally provides a chimeric protein containing ahoming peptide or peptidomimetic which selectively homes to vasculatureof malignant skin and which specifically binds a cognate receptor forCGKRK (SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7) fused to a heterologousprotein. Such a heterologous protein can be, without limitation, aheterologous protein having a therapeutic activity, or an antibody orantigen-binding fragment thereof. In particular embodiments, theinvention provides a chimeric protein in which a homing peptide orpeptidomimetic containing the amino acid sequence CGKRK (SEQ ID NO: 6)or CDTRL (SEQ ID NO: 7) or a conservative variant or peptidomimetic ofone or these sequences, is fused to a heterologous protein.

The term “heterologous,” as used herein in reference to a protein fusedto a homing peptide or peptidomimetic of the invention, means a proteinderived from a source other than the gene encoding the fused homingpeptide or upon which the fused homing peptidomimetic is derived. Achimeric protein of the invention can have a variety of lengthsincluding, but not limited to, up to 100, 200, 300, 400, 500, 800, 1000or 2000 residues or more.

The invention also provides bifunctional peptides. The inventionprovides, for example, a bifunctional peptide which contains a homingpeptide that selectively homes to vasculature of pre-malignantdysplastic skin fused to a second peptide having a separate function,and further provides a bifunctional peptide which contains a homingpeptide that selectively homes to vasculature of malignant skin fused toa second peptide having a separate function. Such bifunctional peptideshave at least two functions conferred by different portions of thepeptide and can, for example, display anti-angiogenic activity orpro-apoptotic activity in addition to selective homing activity. Asnon-limiting examples, the invention provides bifunctional peptides suchas SRPRR-GG-_(D)(KLAKLAK)₂, CSRPPRSEC-GG-_(D)(KLAKLAK)₂,CGKRK-GG-_(D)(KLAKLAK)₂ or CDTRL-GG-_(D)(KLAKLAK)₂. In such peptides,the SRPRR (SEQ ID NO: 1), CSRPPRSEC (SEQ ID NO: 3), CGKRK (SEQ ID NO: 6)or CDTRL (SEQ ID NO: 7) portion exhibits selective homing activity,while the _(D)(KLAKLAK)₂ portion exhibits pro-apoptotic activity.

It is understood that a homing molecule useful in the invention can be,without limitation, a homing peptide or peptidomimetic. As used herein,the term “peptide” is used broadly to mean peptides, proteins, fragmentsof proteins and the like. The term “peptidomimetic,” as used herein,means a peptide-like molecule that has the activity of the peptide uponwhich it is structurally based. Such peptidomimetics include chemicallymodified peptides, peptide-like molecules containing non-naturallyoccurring amino acids, and peptoids, and have an activity such as theselective homing activity of the peptide upon which the peptidomimeticis derived (see, for example, Goodman and Ro, Peptidomimetics for DrugDesign, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed.M. E. Wolff; John Wiley & Sons 1995), pages 803-861).

A variety of peptidomimetics are known in the art including, but notlimited to, peptide-like molecules which contain a constrained aminoacid, a non-peptide component that mimics peptide secondary structure,or an amide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, without limitation, anα-methylated amino acid; α,α-dialkylglycine or α-aminocycloalkanecarboxylic acid; an N^(α)-C^(α) cyclized amino acid; an N^(α)-methylatedamino acid; a β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an N—C^(δ) or C^(α)-C^(δ) cyclizedamino acid; a substituted proline or another amino acid mimetic. Apeptidomimetic which mimics peptide secondary structure can contain,without limitation, a nonpeptidic β-turn mimic; γ-turn mimic; mimic of3-sheet structure; or mimic of helical structure, each of which is wellknown in the art. As non-limiting examples, a peptidomimetic also can bea peptide-like molecule which contains an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylene-sulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptide of theinvention, as well as potential geometrical and chemical complementarityto a target molecule. Where no crystal structure of a peptide of theinvention is available, a structure can be generated using, for example,the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251(1989)). Another database, the Available Chemicals Directory (MolecularDesign Limited, Informations Systems; San Leandro, Calif.), containsabout 100,000 compounds that are commercially available and also can besearched to identify potential peptidomimetics of a peptide of theinvention, for example, with activity in selectively homing topre-malignant dysplastic skin or to malignant skin.

A homing peptide, peptidomimetic or molecule useful in the invention canoptionally be cyclic or otherwise conformationally constrained. As usedherein, a “conformationally constrained” molecule, such as a peptide orpeptidomimetic, is one in which the three-dimensional structure ismaintained substantially in one spatial arrangement over time.Conformationally constrained molecules can have improved properties suchas increased affinity, metabolic stability, membrane permeability orsolubility. Methods of conformational constraint are well known in theart and include, yet are not limited to, cyclization.

As used herein in reference to a peptide or peptidomimetic, the termcyclic refers to a structure including an intramolecular bond betweentwo non-adjacent amino acids or amino acid analogs. The cyclization canbe effected through a covalent or non-covalent bond. Intramolecularbonds include, but are not limited to, backbone to backbone, side-chainto backbone, and side-chain to side-chain bonds. Methods of cyclizationinclude, without limitation, formation of a disulfide bond between theside-chains of non-adjacent amino acids or amino acid analogs; formationof a lactam bond, for example, between a side-chain group of one aminoacid or analog thereof to the N-terminal amine of the amino-terminalamino acid or analog; and formation of lysinonorleucine and dityrosinebonds.

The conjugates and methods of the invention can be practiced with ahoming antibody or antigen-binding fragment thereof which selectivelyhomes to vasculature of pre-malignant dysplastic skin or whichselectively homes to vasculature of malignant skin. As used herein, theterm “antibody” is used in its broadest sense to include polyclonal andmonoclonal antibodies, as well as polypeptide fragments of antibodiesthat retain binding activity for the respective cognate receptor of atleast about 1×10⁵ M⁻¹. One skilled in the art understands that antibodyfragments including, without limitation, Fab, F(ab′)₂ and Fv fragments,can retain binding activity for a cognate receptor and, thus, areincluded within the definition of antibody. In addition, the term“antibody,” as used herein, encompasses non-naturally occurringantibodies and fragments usually containing, at a minimum, one V_(H) andone V_(L) domain, such as chimeric antibodies, humanized antibodies andsingle chain Fv fragments (scFv) that specifically or selectively bindthe appropriate cognate receptor. Such non-naturally occurringantibodies can be constructed using solid phase peptide synthesis,produced recombinantly or obtained by screening phage-displayed or othercombinatorial libraries such as those consisting of variable heavy andlight chains as described in Borrebaeck (Ed.), Antibody Engineering(Second edition) New York: Oxford University Press (1995)) using, forexample, an assay described herein below.

Homing molecules which are antibodies also can be prepared using acognate receptor fusion protein or a synthetic peptide encoding aportion of a cognate receptor. One skilled in the art understands thatpurified human or other cognate receptors, which can be producedrecombinantly, including peptide portions of a cognate receptor such assynthetic peptide fragments can be used as immunogens. It is understoodthat fragments of the cognate receptor for SEQ ID NO: 6 useful asimmunogens include fragments of the cognate receptor that serve toproduce anti-cognate receptor antibodies which are readily internalizedinto cells expressing cell-surface cognate receptor for SEQ ID NO: 6.One skilled in the art further understands that non-immunogenicfragments or synthetic peptides of a cognate receptor for SEQ ID NO: 3,SEQ ID NO: 6 or SEQ ID NO: 7 can be made immunogenic by coupling thehapten to a carrier molecule such as bovine serum albumin (BSA) orkeyhole limpet hemocyanin (KLH). In addition, various other carriermolecules and methods for coupling a hapten to a carrier molecule arewell known in the art as described, for example, by Harlow and Lane,Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press,1988)).

A variety of therapeutic moieties are useful in the conjugates andmethods of the invention, including, without limitation, anti-angiogenicagents and cytotoxic agents, such as those that target a DNA-associatedprocess. As used herein, the term “therapeutic moiety” is used broadlyto mean a physical, chemical, or biological material that can be linkedto a homing molecule and that alters biological activity in a normal orpathologic tissue upon administration. A therapeutic moiety, therefore,is potentially useful for the treatment of disease conditions. Atherapeutic moiety can be any natural or nonnatural material including abiological material, such as a cell or phage; an organic chemical, suchas a small molecule; a radionuclide; a nucleic acid molecule oroligonucleotide; a polypeptide; or a peptide or peptidomimetic.Therapeutic moieties useful in the invention include, withoutlimitation, anti-angiogenic agents; cancer chemotherapeutic agents;cytotoxic agents; pro-apoptotic agents. A therapeutic moiety useful inthe invention can be expressed on, contained in, or linked to any of thefollowing: phage or other virus, cell, liposome, polymeric ornon-polymeric matrix, gold or other particle, or a microdevice,nanodevice, or nano-scale semiconductor material. These and othermaterials known in the art can be components of the conjugates of theinvention.

A therapeutic moiety useful in a conjugate of the invention can be, forexample, an anti-angiogenic agent. As used herein, the term“anti-angiogenic agent” means a molecule that reduces or inhibitsangiogenesis. An anti-angiogenic agent useful in the conjugates andmethods of the invention can be, for example, an inhibitor orneutralizing antibody that reduces the expression or signaling of anangiogenic factor such as vascular endothelial growth factor (VEGF),which is a major inducer of angiogenesis in normal and pathologicalconditions, and is essential in embryonic vasculogenesis. The biologicaleffects of VEGF include stimulation of endothelial cell proliferation,survival, migration and tube formation, and regulation of vascularpermeability. An anti-angiogenic agent also can inhibit anotherangiogenic factor such as a member of the fibroblast growth factor (FGF)family such as FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5 (Slavin etal., Cell Biol. Int. 19:431-444 (1995); Folkman and Shing, J. Biol.Chem. 267:10931-10934 (1992)) or angiopoietin-1, a factor that signalsthrough the endothelial cell-specific Tie2 receptor tyrosine kinase(Davis et al., Cell 87:1161-1169 (1996); and Suri et al., Cell87:1171-1180 (1996)), or the receptor of one of these angiogenicfactors. It is understood that a variety of mechanisms can act toinhibit activity of an angiogenic factor including, without limitation,direct inhibition of receptor binding or of secretion of the angiogenicfactor into the extracellular space, and inhibition of signaling,expression or function of the angiogenic factor.

A variety of anti-angiogenic agents useful in the invention are known inthe art and can be prepared by routine methods. See, for example,Hagedom and Bikfalvi, Crit. Rev. Oncol. Hematol. 34:89-110 (2000) andKirsch et al., J. Neurooncol. 50:149-163 (2000). Anti-angiogenic agentsinclude, without limitation, small molecules; proteins such asangiogenic factors and receptors, transcription factors, and antibodiesand antigen-binding fragments thereof; peptides and peptidomimetics; andnucleic acid molecules including ribozymes, antisense oligonucleotides,and nucleic acid molecules encoding, for example, dominant negativeangiogenic factors and receptors, transcription factors, and antibodiesand antigen-binding fragments thereof. Exemplary anti-angiogenic agentsuseful in the conjugates and methods of the invention include, yet arenot limited to, angiostatin, endostatin, metastatin and 2ME2 (EntreMed;Rockville, Md.); anti-VEGF antibodies such as Avastin (Genentech; SouthSan Francisco, Calif.); VEGFR-2 inhibitors such as the small moleculesSU5416 and SU6668, (SUGEN; South San Francisco, Calif.); heparin-bindingfragments of fibronectin; modified forms of antithrombin; collagenaseinhibitors; basement membrane turnover inhibitors; angiostatic steroids;platelet factor 4, and fragments and peptides thereof; thrombospondin,and fragments and peptides thereof; and doxorubicin (O'Reilly et al.,Cell 79:315-328 (1994)); O'Reilly et al., Cell 88: 277-285 (1997);Homandberg et al., Am. J. Path. 120:327-332 (1985); Biochim. Biophys.Acta 874:61-71 (1986); and O'Reilly et al., Science 285:1926-1928(1999)). It is understood that these as well as other anti-angiogenicagents known in the art or that can be prepared by routine methods areencompassed by the term “anti-angiogenic agent” and can be used in thevarious conjugates and methods of the invention.

It is understood by those skilled in the art that an anti-angiogenicagent can be particularly efficacious when targeted to a specific stageof tumor progression (see, for example, Bergers et al., Science284:808-812 (1999); and Bergers et al., J. Clin. Invest. 111:1287-1295(2003)). Thus, in one embodiment, an anti-angiogenic agent useful in theinvention is “effective against pre-malignant vasculature.” As usedherein, the term “anti-angiogenic agent effective against pre-malignantvasculature” means an angiogenic agent that can significantly reduce thenumber of angiogenic lesions during the pre-malignant phase ofcarcinogenesis, before solid tumors have formed. Such an anti-angiogenicagent is an anti-angiogenic agent effective against pre-malignantvasculature whether or not the agent also significantly reduces tumorburden or extends life-span in animals with tumors, including animalswith small solid tumors or animals having large tumors and end-stagedisease. As non-limiting examples, an anti-angiogenic agent effectiveagainst pre-malignant vasculature can be BB-94 (batimastat), abroad-spectrum inhibitor of matrix metalloproteinases (Talbot and Brown,Eur. J. Cancer 32A: 2528 (1996)); SU5416 (SUGEN), a small moleculeinhibitor of VEGFR-2; or endostatin, a carboxy-terminal fragment ofcollagen XVIII (O'Reilly et al., Cell 88: 277 (1997); and Boehm et al.,Nature 390: 404 (1997)), alone or combined with angiostatin, an internalfragment of plasminogen (O'Reilly et al., Cell 79: 314 (1994); andO'Reilly et al., Nature Med. 2: 689 (1996)). See, also, Bergers et al.,supra, 1999; Bergers et al., supra, 2003.

An anti-angiogenic agent useful in the invention also can be ananti-angiogenic agent effective against tumor vasculature. As usedherein, the term “anti-angiogenic agent effective against tumorvasculature” means an angiogenic agent that can significantly reducetumor burden or extend life-span of animals having solid, vascularizedtumors. Such an anti-angiogenic agent is an anti-angiogenic agenteffective against tumor vasculature if there is efficacy against one ormore of the following: small solid tumors, tumors with well-definedmargins, invasive tumors or end-stage cancer, whether or not the agentsignificantly reduces the number of angiogenic lesions during thepre-malignant phase of carcinogenesis. As non-limiting examples, ananti-angiogenic agent effective against tumor vasculature can beefficacious only against small vascularized tumors, or against largetumors as well as small vascularized tumors. An anti-angiogenic agenteffective against tumor vasculature can be, without limitation, ananti-angiogenic agent such as BB-94 (batimastat), endostatin, orangiostatin, which is effective against small tumors without significantefficacy on the large tumors characteristic of end-stage cancer (Bergerset al., supra, 1999). An anti-angiogenic agent effective against tumorvasculature further can be an anti-angiogenic agent effective againstsmall tumors as well as large tumors in animals with short lifeexpectancy such as, without limitation, AGM-1470 (TNP470), a smallmolecule inhibitor of endothelial cell proliferation (Ingber et al.,Nature 348:555 (1990); Griffith et al., Chem. Biol. 4:461 (1997); Sin etal., Proc. Natl. Acad. Sci. U.S.A. 94:6099 (1997); and Castronovo andBelotti, Eur. J. Cancer 32A:2520 (1996)).

A therapeutic moiety useful in a conjugate of the invention further canbe, for example, a therapeutic moiety with cytotoxicity againstpericytes such as, without limitation, platelet-derived growth factorreceptor-positive pericytes. Such a therapeutic moiety, for example,SU6668, can block further growth of end-stage tumors (Bergers et al.,supra, 2003). Exemplary conjugates include, without limitation,conjugates containing a therapeutic moiety with cytotoxicity againstpericytes linked to a homing peptide or peptidomimetic that selectivelyhomes to vasculature of malignant skin and containing the amino acidsequence CGKRK (SEQ ID NO: 6) or the amino acid sequence CDTRL (SEQ IDNO: 7) or a conservative variant or peptidomimetic thereof.

A therapeutic moiety useful in a conjugate of the invention can be, forexample, a cytotoxic agent. As used herein, the term “cytotoxic agent”means any molecule that results in cell death by any mechanism.Exemplary cytotoxic agents useful in a conjugate of the inventionencompass, without limitation, taxanes such as docetaxel; anthracyclinssuch as doxorubicin; alkylating agents; vinca alkaloids;anti-metabolites; platinum agents such as cisplatin or carboplatin;steroids such as methotrexate; antibiotics such as adriamycin;antimicrobial peptides, described herein below; and other cancerchemotherapeutic agents, which are chemical agents that inhibit theproliferation, growth, life-span or metastatic activity of cancer cells.

Effective cytotoxic agents useful in the invention include those thattarget DNA, for example, alkylating agents, agents that intercalate intoDNA, and agents which result in double-stranded DNA breaks. ExemplaryDNA-targeted drugs include, without limitation, cyclophosphamide,melphalan, mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide,mitoxantrone, SN-38, Et-743, actinomycin D, bleomycin, TLK286 and SGN-15(Hurley, supra, 2002). It is understood that DNA-targeting cytotoxicagents can be particular useful when combined in conjugates with ahoming molecule that localizes, at least in part, to the nuclei ofcells. Thus, in one embodiment the invention provides a conjugatecontaining a therapeutic moiety linked to a homing peptide orpeptidomimetic that selectively homes to vasculature of malignant skinand which specifically binds a cognate receptor for SEQ ID NO: 6, wherethe therapeutic moiety is a cytotoxic agent that targets aDNA-associated process. In a further embodiment, the invention providesa conjugate containing a therapeutic moiety linked to a homing peptideor peptidomimetic which selectively homes to vasculature of malignantskin and which includes the amino acid sequence SEQ ID NO: 6 or aconservative variant or peptidomimetic thereof. Useful cytotoxic agentsthat target a DNA-associated process include, without limitation,alkylating agents, anti-tumor antibiotics and sequence-selective agentsand further encompass agents such as cyclophosphamide, melphalan,mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone,SN-38, Et-743, actinomycin D, bleomycin and TLK286.

Taxanes are cytotoxic agents useful in a conjugate of the invention.Useful taxanes include, without limitation, docetaxel (Taxotere; AventisPharmaceuticals, Inc.; Parsippany, N.J.) and paclitaxel (Taxol;Bristol-Myers Squibb; Princeton, N.J.). See, for example, Chan et al.,J. Clin. Oncol. 17:2341-2354 (1999), and Paridaens et al., J. Clin.Oncol. 18:724 (2000).

A cytotoxic agent useful in a conjugate of the invention also can be ananthracyclin such as doxorubicin, idarubicin or daunorubicin.Doxorubicin is a commonly used cancer chemotherapeutic agent (Stewartand Ratain, In: “Cancer: Principles and practice of oncology” 5th ed.,chap. 19 (eds. DeVita, Jr., et al.; J. P. Lippincott 1997); Harris etal., In “Cancer: Principles and practice of oncology,” supra, 1997). Inaddition, doxorubicin has anti-angiogenic activity which can contributeto its effectiveness in treating cancer (Folkman, supra, 1997; Steiner,In “Angiogenesis: Key principles-Science, technology and medicine,” pp.449-454 (eds. Steiner et al.; Birkhauser Verlag, 1992)).

An alkylating agent such as melphalan or chlorambucil also can be acytotoxic agent useful in a conjugate of the invention. Similarly, avinca alkaloid such as vindesine, vinblastine or vinorelbine; or anantimetabolite such as 5-fluorouracil, 5-fluorouridine or a derivativethereof can be a cytotoxic agent that can be linked to a homing moleculein a conjugate of the invention.

Cytotoxic agents useful in the conjugates of the invention also include,without limitation, platinum agents. Such a platinum agent can be, forexample, cisplatin or carboplatin as described, for example, in Crown,Seminars in Oncol. 28:28-37 (2001). Other cytotoxic agents useful in aconjugate of the invention include, but are not limited to,methotrexate, mitomycin-C, adriamycin, ifosfamide and ansamycins.

A cytotoxic agent also can be, for example, an antimicrobial peptide. Inone embodiment, the invention provides a conjugate in which a homingmolecule that selectively homes to vasculature of pre-malignantdysplastic skin and that specifically binds a cognate receptor for SEQID NO: 3 is linked to an antimicrobial peptide, where the conjugate isselectively internalized by vasculature of a pre-malignant dysplasticlesion, and where the antimicrobial peptide has low mammalian celltoxicity when not linked to the homing molecule. In another embodiment,the invention provides a conjugate in which a homing molecule thatselectively homes to vasculature of malignant skin and that specificallybinds a cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7 is linked toan antimicrobial peptide, where the conjugate is selectivelyinternalized by vasculature of malignant skin, and where theantimicrobial peptide has low mammalian cell toxicity when not linked tothe homing molecule. As used herein, the term “antimicrobial peptide”means a naturally occurring or synthetic peptide having antimicrobialactivity, which is the ability to kill or slow the growth of one or moremicrobes and which has low mammalian cell toxicity when not linked to ahoming molecule. An antimicrobial peptide can kill or slow the growthof, for example, one or more strains of bacteria including aGram-positive or Gram-negative bacteria, or a fingi or protozoa. Thus,an antimicrobial peptide can have, for example, bacteriostatic orbacteriocidal activity against, for example, one or more strains ofEscherichia coli, Pseudomonas aeruginosa or Staphylococcus aureus. Whilenot wishing to be bound by the following, an antimicrobial peptide canhave biological activity due to the ability to form ion channels throughmembrane bilayers as a consequence of self-aggregation.

An antimicrobial peptide is typically highly basic and can have a linearor cyclic structure. As discussed further below, an antimicrobialpeptide can have an amphipathic a-helical structure (see U.S. Pat. No.5,789,542; Javadpour et al., J. Med. Chem. 39:3107-3113 (1996); andBlondelle and Houghten, Biochem. 31: 12688-12694 (1992). Anantimicrobial peptide also can be, for example, a 3-strand/sheet-formingpeptide as described in Mancheno et al., J. Peptide Res. 51:142-148(1998).

An antimicrobial peptide can be a naturally occurring or syntheticpeptide. Naturally occurring antimicrobial peptides have been isolatedfrom biological sources such as bacteria, insects, amphibians, andmammals and are thought to represent inducible defense proteins that canprotect the host organism from bacterial infection. Naturally occurringantimicrobial peptides include the gramicidins, magainins, mellitins,defensins and cecropins (see, for example, Maloy and Kari, Biopolymers37:105-122 (1995); Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994);Bessalle et al., FEBS 274:151-155 (1990); and Blondelle and Houghten inBristol (Ed.), Annual Reports in Medicinal Chemistry pages 159-168Academic Press, San Diego)). As discussed further below, anantimicrobial peptide also can be an analog of a natural peptide,especially one that retains or enhances amphipathicity.

An antimicrobial peptide incorporated within a conjugate of theinvention has low mammalian cell toxicity when not linked to a homingmolecule of the invention. Mammalian cell toxicity readily can beassessed using routine assays. For example, mammalian cell toxicity canbe assayed by lysis of human erythrocytes in vitro as described inJavadpour et al., supra, 1996. An antimicrobial peptide having lowmammalian cell toxicity is not lytic to human erythrocytes or requiresconcentrations of greater than 100 μM for lytic activity, preferablyconcentrations greater than 200, 300, 500 or 1000 μM for lytic activity.

In one embodiment, the invention provides a conjugate in which theantimicrobial peptide portion promotes disruption of mitochondrialmembranes when internalized by eukaryotic cells. In particular, such anantimicrobial peptide preferentially disrupts mitochondrial membranes ascompared to eukaryotic membranes. Mitochondrial membranes, likebacterial membranes but in contrast to eukaryotic plasma membranes, havea high content of negatively charged phospholipids. An antimicrobialpeptide can be assayed for activity in disrupting mitochondrialmembranes using, for example, an assay for mitochondrial swelling oranother assay well known in the art. _(D)(KLAKLAK)₂, for example, is anantimicrobial peptide which induces marked mitochondrial swelling at aconcentration of 10 μM, significantly less than the concentrationrequired to kill eukaryotic cells. An antimicrobial peptide that inducessignificant mitochondrial swelling at, for example, 50 μM, 40 μM, 30 μM,20 μM, 10 μM, or less, is considered a peptide that promotes disruptionof mitochondrial membranes.

An antimicrobial peptide portion can include, for example, the sequence(KLAKLAK)₂ (SEQ ID NO: 13), (KLAKKLA)₂ (SEQ ID NO: 14), (KAAKKAA)₂ (SEQID NO: 15), or (KLGKKLG)₃ (SEQ ID NO: 16), and, in one embodiment,includes the sequence _(D)(KLAKLAK)₂. A conjugate of the invention,which contains a homing molecule that selectively homes to vasculatureof pre-malignant dysplastic skin linked to an antimicrobial peptide, caninclude, for example, the sequence SRPRR-GG-_(D)(KLAKLAK)₂ or thesequence CSRPPRSEC-GG-_(D)(KLAKLAK)₂. Similarly, a conjugate of theinvention containing a homing molecule that selectively homes tovasculature of malignant skin linked to an antimicrobial peptide, caninclude, for example, the sequence CGKRK-GG-_(D)(KLAKLAK)₂ or thesequence CDTRL-GG-_(D)(KLAKLAK)₂.

Antimicrobial peptides generally have random coil conformations indilute aqueous solutions, yet can be induced to high levels of helicitywith helix-promoting solvents and amphipathic media such as micelles,synthetic bilayers or cell membranes. α-Helical structures are wellknown in the art, with an ideal α-helix characterized by having 3.6residues per turn and a translation of 1.5 Å per residue (5.4Å per turn;see Creighton, Proteins: Structures and Molecular Properties W. HFreeman, New York (1984)). In an amphipathic α-helical structure, polarand non-polar amino acid residues are aligned into an amphipathic helix,which is an α-helix in which the hydrophobic amino acid residues arepredominantly on one face, with hydrophilic residues predominantly onthe opposite face when the peptide is viewed along the helical axis.

Antimicrobial peptides of widely varying sequence have been isolated,sharing an amphipathic α-helical structure as a common feature (Saberwalet al., Biochim. Biophys. Acta 1197:109-131 (1994)). Analogs of nativepeptides with amino acid substitutions predicted to enhanceamphipathicity and helicity typically have increased antimicrobialactivity. In general, analogs with increased antimicrobial activity alsohave increased cytotoxicity against mammalian cells (Maloy et al.,Biopolymers 37:105-122 (1995)). Synthetic antimicrobial peptides havingan amphipathic α-helical structure are known in the art as described,for example, in U.S. Pat. No. 5,789,542 to McLaughlin and Becker.

It is understood by one skilled in the art of medicinal oncology thatthese and other agents are useful therapeutic moieties, which can beused separately or together in the conjugates and methods of theinvention. It further is understood that a conjugate of the inventioncan contain one or more of such therapeutic moieties and that additionalcomponents can be included as part of the conjugate, if desired. As anexample, in some cases it can be desirable to utilize an oligopeptidespacer between the homing molecule and the therapeutic agent(Fitzpatrick and Garnett, Anticancer Drug Des. 10:1-9 (1995)).

In further embodiments, a conjugate of the invention includes adetectable moiety. As used herein, the term “detectable moiety” meansany molecule which can be administered in vivo and subsequentlydetected. Exemplary detectable moieties useful in the conjugates andmethods of the invention include, without limitation, radiolabels andfluorescent molecules. Exemplary radionuclides include indium-111,technetium-99, carbon-11, and carbon-13. Fluorescent molecules include,without limitation, fluorescein, allophycocyanin, phycoerythrin,rhodamine, and Texas red.

The methods of the invention for imaging the vasculature of apre-malignant dysplastic tissue such as dysplastic skin or thevasculature of a malignant tissue can be useful for early detection ofdysplastic lesions or malignancies including but not limited to,dermatological dysplasias and malignancies. Following administration ofa conjugate of the invention containing a detectable moiety, thevasculature of pre-malignant dysplastic tissue or malignant tissue isvisualized. If the image is positive for the presence of suchvasculature, further evaluation can be performed for the size of thetumor, if any, and the quantity of vascular infiltration. These resultsprovide valuable information to the clinician with regard to the stageof development of the cancer and the presence or probability ofmetastasis.

It is understood that the methods of the invention are applicable to avariety of types of pre-malignancies and malignancies including, yet notlimited to, squamous cell dysplasias and carcinomas, melanomas and otherdermatological dysplasias and malignancies; breast dysplasias andmalignancies; ovarian dysplasias and malignancies; cervical dysplasiasand malignancies; prostate dysplasias and malignancies; lung dysplasiasand malignancies; and other epitheliomas, epithelial cell dysplasias andmalignancies.

The methods of the invention are applicable to a variety of neoplasms ofepithelial origin ranging from benign to malignant and includingdermatological and non-dermatological dysplasias and malignancies. Asnon-limiting examples, the methods of the invention are applicable to avariety of types of dermatological dysplasias and malignancies such assquamous cell carcinomas, basosquamous cell carcinomas, basal cellcarcinomas, cancer en cuirasse and Merkel cell carcinomas. As furthernon-limiting examples, the methods of the invention are applicable to,without limitation, breast carcinomas including apocrine cellcarcinomas; acinar cell carcinomas such as of the breast or salivarygland; ductal carcinomas such as those of the pancreas or breast;endometrial carcinomas; alveolar and bronchiogenic carcinomas includingadenocarcinomas of the lung, large cell carcinomas, small cellcarcinomas (small cell lung cancers) and squamous cell carcinomas; giantcell carcinomas, for example, of the lung or thyroid gland; gastriccarcinomas such as adenocarcinomas and carcinoid tumors of the small orlarge intestine; schistosomal bladder carcinomas; intra- andextrahepatic bile duct carcinomas; hepatocellular carcinomas (hepatomas,hepatocarcinomas); renal cell carcinomas; thyroid gland carcinomas;epithelial tumors of the salivary gland; adenocystic and adenoidsquamous cell carcinomas, adenomas; nasopharyngeal carcinomas; meningealcarcinomas; and embryonal carcinomas. One skilled in the art understandsthat the methods of the invention, including but not limited to methodsof directing a moiety to vasculature of a malignant tissue, can beapplied to any of the above and other epithelial dysplasias andmalignancies. In particular embodiments, the methods of the inventionrely on a conjugate containing a moiety linked to a homing molecule thatselectively homes to vasculature of malignant skin and whichspecifically binds a cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7in order to direct a moiety, image vasculature or treat one of theabove-specified dysplasias or malignancies.

In a method of the invention for imaging vasculature of a pre-malignantdysplastic tissue, the conjugate administered contains a detectablemoiety that allows detection or visualization of the vasculature of thepre-malignant dysplastic tissue such as dysplastic skin. In a method ofthe invention for imaging vasculature of a malignant tissue, theconjugate administered contains a detectable moiety that allowsdetection or visualization of the vasculature of the malignant tissuesuch as malignant skin. For such in vivo diagnostic imaging, a homingmolecule is linked to a detectable moiety that, upon administration tothe subject, is detectable external to the subject. Such a detectablemoiety can be, for example, a gamma ray emitting radionuclide such asindium-113, indium-115 or technetium-99; following administration to asubject, the conjugate can be visualized using a solid scintillationdetector.

It is understood that a variety of routes of administration are usefulin the methods of the invention. Such routes encompass systemic andlocal administration and include, without limitation, oraladministration, topical application, intravenous injection,intraperitoneal injection, intramuscular injection, subcutaneousinjection, transdermal diffusion or electrophoresis, local injection,and extended release delivery devices including locally implantedextended release devices such as bioerodible or reservoir-basedimplants.

As disclosed herein, several homing peptides identified as selectivelyhoming to vasculature of pre-malignant dysplastic skin such as homingpeptides SEQ ID NOS: 3, 4 and 5 share the sequence SRPRR (SEQ ID NO: 1),present in kallikrein 9. Thus, a cognate receptor for a homing moleculethat selectively homes to vasculature of pre-malignant dysplastic skincan be a molecule that specifically binds a kallikrein 9 bindingmolecule such as a kallikrein 9 substrate or inhibitor. Based on theresults disclosed herein, the present invention provides a method ofisolating one or more homing molecules that selectively home tovasculature of pre-malignant dysplastic skin by contacting a kallikrein9 binding molecule, or a fragment thereof, with a library of moleculesunder conditions suitable for specific binding of a molecule to thekallikrein 9 binding molecule; assaying for specific binding; andseparating from the library one or more molecules that specifically bindthe kallikrein 9-binding molecule, thereby isolating one or more homingmolecules that selectively home to vasculature of pre-malignantdysplastic skin.

As non-limiting examples, native, recombinant and human kallikrein9-binding molecules, and fragments of a kallikrein 9-binding moleculeincluding fragments that bind SEQ ID NOS: 3, 4 or 5, whether purified orexpressed on the surface of a cell, can be useful in the screeningmethods of the invention. In one embodiment, the kallikrein 9-bindingmolecule is a kallikrein 9 inhibitor. In another embodiment, thekallikrein 9-binding molecule is an isolated kallikrein 9 bindingmolecule. In a further embodiment, the kallikrein 9-binding molecule iscell-surface expressed molecule. It is understood that libraries thatcan be screened according to a method of the invention include, but arenot limited to, libraries of peptides and peptidomimetics, libraries ofsmall molecules, and libraries of antibodies and antigen-bindingfragments thereof, including synthetic, single-chain or other antibodylibraries.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Phage that Selectively Home to Dysplastic Skin

This example describes identification of a peptide that selectivelyhomes to dysplastic skin lesions.

To isolate peptides specific for dysplastic lesions, two rounds ofselection were performed ex vivo followed by one round of selection invivo. Ex vivo and in vivo phage selections were performed with anNNK-encoded CX₇C (SEQ ID NO: 8) peptide library displaying 9-mer cyclicpeptides with seven degenerate positions on Novagen's T7 415-1b phagevector; the library had a diversity of approximately 1×10⁸. Phageselections and validations were performed essentially as described inLaakkonen et al., Nat. Med. 8:751-755 (2002).

For ex vivo selections, phage were incubated with dispersed cells fromdysplastic skin removed from the ears and chest of K14-HPV16 mice withno macroscopic evidence of tumors at 4-6 months of age. The dysplasticskin lesions typically included a focal region of epidermal dysplasiaflanked by adjacent hyperplastic epidermis, along with underlyingreactive stromal elements and angiogenic endothelium in the aberrantdermis. A portion of each dysplastic skin tissue biopsy used for ex vivoor in vivo phage selection was fixed in formalin and, after conventionalprocessing, examined histologically to assess the neoplastic grade.Hematoxylin and eosin stained paraffin sections confirmed that the areasof skin used in the selection steps were largely made up of focaldysplasias.

Sequential ex vivo selections on cell suspensions of dysplastic skinresulted in a 160-fold enrichment of phage relative to similar treatmentwith non-recombinant phage lacking displayed peptides (FIG. 1A, left);greater than 10,000-fold enrichment resulted from the subsequent roundof in vivo selection (see FIG. 1A, right). Selection in vivo alsoproduced a minor enrichment of phage that homed to control brain, kidneyand hyperplastic skin tissues. DNA sequence analysis of 48 clonesisolated in the second ex vivo round and 48 clones from the subsequentin vivo round characterized the sequences of the CX₇C (SEQ ID NO: 8)homing peptides. Of the 96 phage clones analyzed, nine peptides appearedmost frequently and were further analyzed as described below.

To test homing specificity of the selected peptides, purified phagedisplaying a single recombinant sequence were intravenously injectedinto K14HPV16 mice bearing protuberant ear or trunk tumors at 9 to 12months of age to assess homing to squamous cell carcinomas, oralternatively injected into younger K14HPV16 mice presenting withmultifocal dysplasias but no tumors. Both neoplastic tissues and normalcontrol organs were collected and assayed for phage accumulation. Phagedisplaying the peptide CRAKSKVAC (SEQ ID NO: 9), which appeared with thehighest frequency in both the ex vivo and in vivo sequence pools (5 and15 times, respectively), were 1500-fold enriched in dysplastic skinrelative to non-recombinant control phage. However, phage displayingpeptide SEQ ID NO: 9 accumulated with similar frequency in normal skin,kidney and brain, indicating that the CRAKSKVAC (SEQ ID NO: 9) peptidehomes to an abundant signal in each of these organs and is not specificto dysplastic skin lesions. Several other dysplasia-selected clones alsodifferentiated poorly between control tissues and dysplastic skin; sevenof the nine dysplasia-selected phage clones appeared to be responsiblefor the overall enrichment of recombinant phage in control tissues inthe final in vivo round of selection as shown in FIG. 1A (right panel),and were not studied further.

Peptides displayed on the two remaining phage clones were found to behighly selective for dysplastic skin, and did not appreciably home tonormal organs. One of these peptides, CNRRTKAGC (SEQ ID NO: 10), isclosely related to a previously described peptide that homes to tumorlymphatic vessels (Laakkonen, supra, 2002). The second dysplasia-homingpeptide, CSRPRRSEC (SEQ ID NO: 3), appeared three times amongst the 48phage sequenced from the in vivo round, along with two variants,CSRPRRSVC (SEQ ID NO: 4) and CSRPRRSWC (SEQ ID NO: 5), that eachappeared once. Phage displaying the CSRPRRSEC (SEQ ID NO: 3) peptidewere enriched ˜350-fold in dysplastic skin and did not significantlyaccumulate in control tissues (FIG. 1C, left panel). Furthermore, wheninjected into a K14HPV16 mouse bearing an ear tumor as well asmultifocal skin dysplasias, CSRPRRSEC (SEQ ID NO: 3)-displaying phageeffectively homed to dysplastic chest skin and dysplastic ear skin, butshowed little homing to the tumor (FIG. 1C, right panel). In addition,CSRPRRSEC (SEQ ID NO: 3)-bearing phage did not home to normal skin ofFVB/n mice in vivo and further did not bind to hyperplastic skin of 1 to2 month-old K14-HPV16 mice in ex vivo experiments (FIG. 1C, left andright panels).

These results demonstrate that the CSRPRRSEC (SEQ ID NO: 3) peptideselectively homes to dysplastic skin lesions and further indicate thatthis peptide binds to a receptor which is present in pre-malignant skindysplasias but which is essentially absent or inaccessible via thecirculation in normal skin and in skin malignancies such as squamouscell carcinomas. Given that peptide CSRPRRSEC (SEQ ID NO: 3) localizesto endothelial cells, these results further indicate that homingselectivity is attributable to vascular changes during the carcinogenicprogression from normal skin to dysplasia and then to cancer.

EXAMPLE II Tumor-Specific Homing Phage

This example describes identification of phage that selectively home tomalignant skin such as squamous cell carcinomas.

To isolate phage that selectively home to squamous cell carcinomas(SCCs), two rounds of ex vivo panning selections were performed followedby two rounds of in vivo panning in K14-HPV16 mice having tumorshistologically confirmed as squamous cell carcinoma grades II-IV(Coussens, supra, 1996). The enrichment rose from 6-fold relative tonon-recombinant phage in the second ex vivo round to greater than70-fold relative to non-recombinant phage in the second in vivo round(the fourth sequential round overall) as shown in FIG. 1B. From 192sequenced phage clones, fifteen were selected for further analysis basedon their frequency (48 from ex vivo round 2, 48 from in vivo round 1,and 96 from in vivo round 2) and their increased prevalence in the invivo selections. Of these, four clones displaying the following aminoacid sequences bound to a K14-HPV16 tumor-derived cell suspension exvivo: CGKRK (SEQ ID NO: 6), CGTKRKC (SEQ ID NO: 11), CDTAVVEGL (SEQ IDNO: 12) and CDTRL (SEQ ID NO: 7). Phage-displaying the sequenceCDTAVVEGL (SEQ ID NO: 12) also were 340-fold enriched in tumors in vivorelative to non-recombinant control phage.

When intravenously injected into tumor-bearing K14-HPV16 mice, CGKRK(SEQ ID NO: 6) phage showed a marked preference for the tumor, with anefficiency that varied from 80 to 1,000-fold in independent experiments.Some homing to dysplastic lesions was observed in the one of twoexperiments that showed an 80× enrichment in the tumor (FIG. 1D). Normaland hyperplastic skin, and various control organs, accumulated very lowlevels of the CGKRK (SEQ ID NO: 6) phage. Similar analysis of the CDTRL(SEQ ID NO: 7) phage revealed a variable preference for squamous cellcarcinomas and dysplastic lesions; in one experiment the phageaccumulated more effectively in dysplastic lesions than in a tumor,whereas the reverse was true in another experiment (FIG. 1E). This phageshowed little affinity for hyperplastic skin, and no significant homingto normal skin from FVB/n mice (FIG. 1E). Thus, phage displaying peptideCDTRL (SEQ ID NO: 7) were variably selective for both dysplasias andsquamous tumors of the epidermis, indicative of lesional heterogeneityin the CDTRL (SEQ ID NO: 7) receptor.

EXAMPLE III Intra-Tissue Localization of Homing Peptides

This example describes localization of several homing peptides.

To characterize the nature of the selectivity of the dysplasia- andtumor-homing peptides, K14-HPV16 mice were intravenously injected withcloned phage displaying a particular peptide, and phage localizationvisualized using histological procedures. Peptide localization wasfurther analyzed with chemically synthesized fluorescein-labeledpeptides.

Phage were intravenously injected into 4 to 6 month-olddysplasia-bearing mice with dysplasia-homing phage bearing CSRPRRSEC(SEQ ID NO: 3). In parallel, a set of 9 to 12 month-old tumor-bearingmice were infused with one of the tumor-homing phage CGTKRKC (SEQ ID NO:11), CGKRK (SEQ ID NO: 6) or CDTRL (SEQ ID NO: 7). Various tissues werecollected from each mouse and analyzed by double labelimmunohistochemical staining using an anti-T7 antibody to detect phageand an anti-CD31 antibody to detect endothelial cells of thevasculature. As shown in FIG. 2, phage co-localized in each case withCD31-positive endothelial cells in the expected target tissue. Inparticular, CSRPRRSEC (SEQ ID NO: 3)-phage accumulated in dysplasticskin (FIG. 2A) of dysplastic mice, CGTKRKC (SEQ ID NO: 11) phage werealso detected to a lesser extent in dysplastic skin of tumor-bearingmice (FIG. 2B), CGKRK (SEQ ID NO: 6) phage accumulated in tumor tissue(FIG. 2C), and CDTRL (SEQ ID NO: 7) phage were localized to large,dilated vessels throughout the dysplastic and hyperplastic skin (FIG.2D) as well as in tumors (FIG. 2E).

To further evaluate homing selectivity of displayed peptides, syntheticpeptides were analyzed outside of the context of phage particles. Bothyounger dysplasia-bearing and older tumor-bearing K14-HPV16 mice wereinjected with fluorescein-labeled peptides. After 10 minutes, bothnormal and neoplastic tissues were collected; tissue sections wereprepared and stained with antibodies to both CD31 and a secondendothelial marker, the cell-surface antigen Meca-32. Similar resultswere obtained with the two endothelial marker antibodies; the data forMeca-32 are shown in FIG. 3, in which localization of intravenouslyinfused peptides and antibody was visualized by two-color fluorescencemicroscopy.

As shown in FIG. 3, fluorescein-labeled peptides co-localized withMeca-32 in target neoplastic tissue after intravenous injection, andwere not detected in tissues where the corresponding phage did not home.Specifically, fluorescein-labeled CSRPRRSEC (SEQ ID NO: 3) co-localizedwith Meca-32 in dysplastic skin vasculature from both non-tumor-bearing(FIG. 3A) and tumor-bearing mice (FIG. 3D, inset); notably, the peptidewas not detected within the squamous tumor in the latter (FIG. 3D),confirming its selectivity for premalignant dysplastic vasculature. Incontrast, fluorescein-labeled CGKRK (SEQ ID NO: 6) and CDTRL (SEQ ID NO:7) peptides were not detected in the dysplastic skin of youngernon-tumor bearing mice (FIGS. 3B and 3C) but were primarily detected intumor vasculature (FIGS. 3E and F), and at lower levels in thedysplastic skin of these tumor-bearing mice. Together with theimmunolocalization analyses of phage homing, the peptide localizationdata indicate that CGKRK (SEQ ID NO: 6) and CDTRL (SEQ ID NO: 7) homespecifically to blood vessels in squamous cell carcinomas and in thedysplastic foci of tumor-bearing mice, but not to vasculature of earlierstage dysplasias in non-tumor bearing mice.

Areas of skin, tumors and various organs were collected and either fixedin formalin, dehydrated through serial alcohols and embedded inparaffin, or directly embedded in OCT medium (Fisher Scientific).K14-HPV16 dysplasia and tumor samples were graded by evaluatinghematoxylin & eosin and anti-keratin staining on 5 μm paraffin sectionsunder a light microscope (Coussens et al., supra, 1996). Rat anti-mouseCD31 and rat anti-mouse Meca-32 (BD Pharmingen; San Diego, Calif.) wereused for vascular immunostaining on 10 μm frozen sections. Anti-phagestaining and biodistribution of fluorescein-labeled peptides wereperformed essentially as described in Laakkonen et al., supra, 2002.

EXAMPLE IV Tumor-Type Specificity of Peptide Homing

This example describes tumor-type specificity of peptide homing.

Peptides identified by their binding to endothelial cells in skindysplasias or skin tumors could in principle be selective for neoplasiasin this tissue, neoplasias of this cell type, neoplasias induced bythese oncogenes, or be general to neoplasias in various tissues and ofvarious cell types and oncogenic transformations.

To analyze homing selectivity, the K14-HPV16 squamous cell cancer-homingpeptides, CGKRK (SEQ ID NO: 6) and CDTRL (SEQ ID NO: 7), were analyzedfor the ability to home to endothelium in tumors of different tissueorigins and in tumors localized to different anatomical locations. Inparticular, three subcutaneously implanted tumors and two tumorsproduced in transgenic animal models were examined for accumulation offluorescein-labeled peptides following intravenous injection

As shown in FIG. 4, different homing specificities were observed foreach peptide in the various tumor microenvironments. In particular, inFIGS. 4A and F, neither peptide CGKRK (SEQ ID NO: 6) nor CDTRL (SEQ IDNO: 7) homed to angiogenic islets (dysplasias) or tumors in the RIP-Tagtransgenic mouse model of pancreatic islet cell carcinoma (Hanahan,Nature 315:115-122 (1985)), indicating that the binding moieties forthese peptides are not present in normal, dysplastic, or pancreatictumor vasculature. However, both CGKRK (SEQ ID NO: 6) and CDTRL (SEQ IDNO: 7) did home to breast carcinomas in the MMTV-PyMT transgenic mousemodel (Guy et al., Mol. Cell. Biol. 12:954-961 (1992)) as shown in FIGS.4B and G Some of the positive cells appeared to becirculation-accessible tumor cells (Chang et al., Proc. Natl. Acad. Sci.USA 97:14608-14613 (2000)), indicating that the CGKRK (SEQ ID NO: 6) andCDTRL (SEQ ID NO: 7) peptide binding receptors can have broaderrepresentation beyond tumor endothelial cells (see FIG. 4G). Both CGKRK(SEQ ID NO: 6) and CDTRL (SEQ ID NO: 7) bind a range of cultured tumorcells in addition to homing to tumor endothelial cells in vivo. Further,the activated endothelium in the MMTV-PyMT mouse breast tumor modelshares molecular determinants with squamous cell carcinomas of the skinas detected by these peptides; these determinants are not present in theRIP-Tag model of endocrine pancreatic cancer. Skin squamous cellcarcinoma tumorigenesis is induced by the E6 and E7 oncogenes of HPV16,while both RIP-Tag and MMTV-PyMT tumors are induced by the polyomamiddle T-antigen, indicating that homing selectivity is not dependent ontransformation by a particular oncogene or oncogenes.

The two tumor peptides showed different homing specificity when assayedfor the ability to home to three types of subcutaneously growntransplanted tumors. Fluorescein-CGKRK (SEQ ID NO: 6) peptide homed tocells in each of the three transplant tumors (FIGS. 4C-E), which arosefrom PDSC5, a K14-HPV16 tumor-derived cell line (FIG. 4C); theMDA-MB-435 human breast cancer line (FIG. 4D; Price et al., Cancer Res.50:717-721 (1990)); or the C8161 human melanoma line (FIG. 4E; Bregmanand Meyskens, Int. J. Cancer 37:101-107 (1986)). In contrast, the CDTRL(SEQ ID NO: 7) peptide accumulated only in the melanoma xenografts (FIG.4J) and in the skin overlying the melanoma xenograft tumor (FIG. 4Jinset). Furthermore, fluorescein-CGKRK (SEQ ID NO: 6) localized in thecytoplasm and nuclei of vascular cells identified as endothelial cellsby their morphology and by immunostaining for CD31 and Meca-32 (see FIG.4D). In addition, peptide CGKRK (SEQ ID NO: 6) apparently extravasatedout of the vessels and distributed along tendril-like structures and intumor cell nuclei; the peptide also accumulated to some extent inavascular necrotic regions (FIGS. 4B, C and E). Given that all threetumors were growing subcutaneously, by presumably recruitingneovasculature from the same normal vascular bed, these results indicatethat cell type or oncogenic stimulus imparts different qualities ontovasculature and the tumor microenvironment, as revealed by differentialselective homing patterns seen with these peptides.

RIP1-Tag2 mice were used at 12 weeks of age, at which time all mice hadpancreatic islet tumors. MMTV-PyMT mice were 4 to 6 months old andcarried palpable mammary tumors. Cells for subcutaneous inoculation toproduce transplant tumors were cultured in 10% fetal calf serum (FCS) inDulbecco's Modified Eagle's Media (DMEM). Tumors were generated bysubcutaneously injecting 10⁶ cells into the chest skin of FVB/n (PDSC5)or Balb/c nude (MDA-MB-435 and C8161) mice. Mice bearing subcutaneoustransplant tumors had tumors with an approximate diameter of 1centimeter. PDSC5 tumors and MDA-MB-435 xenografts reached this sizeabout 9 weeks post-injection while C8161 xenografts reached a size of 1cm about three weeks post-injection.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. A conjugate, comprising a therapeutic moiety linked to a homingpeptide or peptidomimetic that selectively homes to vasculature ofpre-malignant dysplastic skin, said homing peptide or peptidomimeticcomprising the amino acid sequence SRPRR (SEQ ID NO: 1) the amino acidsequence CGKRK (SEQ ID NO: 6) or the amino acid sequence CDTRL (SEQ IDNO: 7) or a conservative variant or peptidomimetic thereof.
 2. Theconjugate of claim 1, wherein the peptide or peptidomimetic portion ofsaid conjugate has a length of at most 200 residues.
 3. The conjugate ofclaim 1, wherein the peptide or peptidomimetic portion of said conjugatehas a length of at most 50 residues.
 4. The conjugate of claim 1,wherein said homing peptide or peptidomimetic is conformationallyconstrained.
 5. The conjugate of claim 4, wherein said conformationallyconstrained homing peptide or peptidomimetic is cyclic. 6-7. (canceled)8. The conjugate of claim 1, wherein said homing peptide orpeptidomimetic comprises the amino acid sequence CXSRPRRZC (SEQ ID NO:2) or a conservative variant or peptidomimetic thereof, wherein X=0 to20 independently selected residues and wherein Z=0 to 20 independentlyselected residues. 9-10. (canceled)
 11. The conjugate of claim 8,wherein said homing peptide or peptidomimetic comprises the amino acidsequence CSRPRRSEC (SEQ ID NO: 3) or a conservative variant orpeptidomimetic thereof. 12-13. (canceled)
 14. The conjugate of claim 8,wherein said homing peptide or peptidomimetic comprises the amino acidsequence CSRPRRSVC (SEQ ID NO: 4), or a conservative variant orpeptidomimetic thereof. 15-16. (canceled)
 17. The conjugate of claim 8,wherein said homing peptide or peptidomimetic comprises the amino acidsequence CSRPRRSWC (SEQ ID NO: 5), or a conservative variant orpeptidomimetic thereof. 18-19. (canceled)
 20. The conjugate of claim 1,8, 12, 15 or 18, wherein said therapeutic moiety is an anti-angiogenicagent.
 21. The conjugate of claim 20, wherein said anti-angiogenic agentis effective against pre-malignant vasculature or tumor vasculature. 22.The conjugate of claim 1, 8, 12, 15 or 18, wherein said therapeuticmoiety is a cytotoxic agent.
 23. A method of directing a moiety tovasculature of a pre-malignant dysplastic tissue in a subject,comprising administering to the subject a conjugate which comprises amoiety linked to a homing molecule that selectively homes to vasculatureof pre-malignant dysplastic skin, said homing molecule specificallybinding a cognate receptor for CSRPRRSEC (SEQ ID NO: 3), therebydirecting the moiety to vasculature of said pre-malignant dysplastictissue.
 24. The method of claim 23, wherein said homing molecule is notan antibody or antigen-binding fragment thereof.
 25. The method of claim23, wherein said homing molecule is a kallikrein 9 analog.
 26. Themethod of claim 23, wherein said homing molecule is a homing peptide orpeptidomimetic.
 27. The method of claim 26, wherein the peptide orpeptidomimetic portion of said conjugate has a length of at most 200residues.
 28. The method of claim 26, wherein the peptide orpeptidomimetic portion of said conjugate has a length of at most 50residues.
 29. The method of claim 26, wherein said homing peptide orpeptidomimetic comprises the amino acid sequence SRPRR (SEQ ID NO: 1) ora conservative variant or peptidomimetic thereof.
 30. The method ofclaim 29, wherein said homing peptide or peptidomimetic isconformationally constrained.
 31. The method of claim 30, wherein saidconformationally constrained homing peptide or peptidomimetic is cyclic.32-33. (canceled)
 34. The method of claim 26, wherein said homingpeptide or peptidomimetic comprises the amino acid sequence CXSRPRRZC(SEQ ID NO: 2) or a conservative variant or peptidomimetic thereof,wherein X=0 to 20 independently selected residues and wherein Z=0 to 20independently selected residues.
 35. The method of claim 34, whereinsaid homing peptide or peptidomimetic comprises the amino acid sequenceCSRPRRSEC (SEQ ID NO: 3) or a conservative variant or peptidomimeticthereof.
 36. The method of claim 34, wherein said homing peptide orpeptidomimetic comprises the amino acid sequence CSRPRRSVC (SEQ ID NO:4), or a conservative variant or peptidomimetic thereof.
 37. The methodof claim 34, wherein said homing peptide or peptidomimetic comprises theamino acid sequence CSRPRRSWC (SEQ ID NO: 5), or a conservative variantor peptidomimetic thereof.
 38. The method of claim 23, wherein saidmoiety is a therapeutic moiety.
 39. The method of claim 38, wherein saidtherapeutic moiety is an anti-angiogenic agent.
 40. The method of claim39, wherein said anti-angiogenic agent is effective againstpre-malignant vasculature.
 41. The method of claim 38, wherein saidtherapeutic moiety is a cytotoxic agent.
 42. The method of claim 23,wherein said moiety is a detectable moiety.
 43. The method of claim 42,wherein said detectable moiety is a radionuclide.
 44. The method ofclaim 43, wherein said radionuclide is selected from the groupindium-111, technetium-99, carbon-11, and carbon-13.
 45. The method ofclaim 42, wherein said detectable moiety is a fluorescent label.
 46. Amethod of imaging vasculature of a pre-malignant dysplastic tissue,comprising the steps of: a) administering to the subject a conjugatecomprising a detectable moiety linked to a homing molecule thatselectively homes to vasculature of pre-malignant dysplastic skin, saidhoming molecule specifically binding a cognate receptor for CSRPRRSEC(SEQ ID NO: 3), b) and detecting said conjugate, thereby imagingvasculature of said pre-malignant dysplastic tissue. 47-61. (canceled)62. A method of reducing the risk of progression to a malignancy in asubject, comprising administering to the subject a conjugate whichcomprises a therapeutic moiety linked to a homing molecule thatselectively homes to vasculature of pre-malignant dysplastic skin, saidhoming molecule specifically binding a cognate receptor for CSRPRRSEC(SEQ ID NO: 3), thereby diminishing vasculature of a pre-malignantdysplastic tissue and reducing the risk of progression to malignancy.63-91. (canceled)
 92. The conjugate of claim 1, wherein said therapeuticmoiety is a cytotoxic agent that targets a DNA-associated process. 93.The conjugate of claim 92, wherein said cytotoxic agent is selected fromthe group an alkylating agent, an anti-tumor antibiotic and asequence-selective agent.
 94. The conjugate of claim 92, wherein saidcytotoxic agent is selected from the group cyclophosphamide, melphalan,mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone,SN-38, Et-743, actinomycin D, bleomycin and TLK286.
 95. A method ofdirecting a moiety to vasculature of a malignant tissue in a subject,comprising administering to the subject a conjugate which comprises amoiety linked to a homing molecule that selectively homes to vasculatureof malignant skin, said homing molecule specifically binding a cognatereceptor for SEQ ID NO: 6 or SEQ ID NO: 7, thereby directing the moietyto vasculature of said malignant tissue. 96-111. (canceled)
 112. Amethod of imaging vasculature of a malignant tissue, comprising thesteps of: a) administering to the subject a conjugate comprising adetectable moiety linked to a homing molecule that selectively homes tovasculature of malignant skin, said homing molecule specifically bindinga cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7 and b) detectingsaid conjugate, thereby imaging vasculature of said malignant tissue.113-129. (canceled)
 130. A method of treating a cancer in a subject,comprising administering to the subject a conjugate which comprises atherapeutic moiety linked to a homing molecule that selectively homes tovasculature of malignant skin, said homing molecule specifically bindinga cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7, thereby directingsaid therapeutic moiety to vasculature of said cancer and treating saidcancer. 131-150. (canceled)
 151. A method of staging tumor progressionin a subject having or suspected of having a premalignant lesion ortumor, comprising: a) administering to said subject a conjugatecomprising a detectable moiety linked to (i) a homing molecule thatselectively homes to vasculature of pre-malignant dysplastic skin, saidhoming molecule specifically binding a cognate receptor for CSRPRRSEC(SEQ ID NO: 3), or (ii) a homing molecule that selectively homes tovasculature of malignant skin, said homing molecule specifically bindinga cognate receptor for SEQ ID NO: 6 or SEQ ID NO: 7; and b) detectingsaid conjugate, wherein detection of said conjugate comprising a homingmolecule that selectively homes to vasculature of pre-malignantdysplastic skin indicates a pre-malignant stage of tumor progression insaid subject and wherein detection of said conjugate comprising a homingmolecule that selectively homes to vasculature of malignant skinindicates a malignant stage of tumor progression in said subject.152-170. (canceled)
 171. An isolated peptide or peptidomimetic, having alength of less than 20 residues and comprising the amino acid sequenceselected from SRPRR (SEQ ID NO: CGKRK (SEQ ID NO: 6) and CDTRL (SEQ IDNO: 7) or a peptidomimetic thereof.
 172. The isolated peptide orpeptidomimetic of claim 171, which is a peptide.
 173. The isolatedpeptide or peptidomimetic of claim 171, which is conformationallyconstrained.
 174. The isolated peptide or peptidomimetic of claim 173,which is cyclic.
 175. The isolated peptide or peptidomimetic of claim171, comprising the amino acid sequence CSRPRRSEC (SEQ ID NO: 3) or apeptidomimetic thereof.
 176. The isolated peptide or peptidomimetic ofclaim 171, comprising the amino acid sequence CSRPRRSVC (SEQ ID NO: 4)or a peptidomimetic thereof.
 177. The isolated peptide or peptidomimeticof claim 171, comprising the amino acid sequence CSRPRRSWC (SEQ ID NO:5) or a peptidomimetic thereof.
 178. The isolated peptide orpeptidomimetic of claim 171 or 173, having a length of less than 15residues.
 179. The isolated peptide or peptidomimetic of claim 171 or173, having a length of less than 10 residues.
 180. An isolated peptideor peptidomimetic, having a length of less than 90 residues andcomprising the amino acid sequence CXSRPRRZC (SEQ ID NO: 2) or apeptidomimetic thereof, wherein X=0 to 20 independently selectedresidues and wherein Z=0 to 20 independently selected residues.
 181. Theisolated peptide or peptidomimetic of claim 180, which is a peptide.182. The isolated peptide or peptidomimetic of claim 180, which isconformationally constrained.
 183. The isolated peptide orpeptidomimetic of claim 182, which is cyclic.
 184. The isolated peptideor peptidomimetic of claim 180, comprising the amino acid sequenceCSRPRRSEC (SEQ ID NO: 3) or a peptidomimetic thereof.
 185. The isolatedpeptide or peptidomimetic of claim 180, comprising the amino acidsequence CSRPRRSVC (SEQ ID NO: 4) or a peptidomimetic thereof.
 186. Theisolated peptide or peptidomimetic of claim 180, comprising the aminoacid sequence CSRPRRSWC (SEQ ID NO: 5) or a peptidomimetic thereof. 187.The isolated peptide or peptidomimetic of claim 180, having a length ofless than 60 residues.
 188. The isolated peptide or peptidomimetic ofclaim 180, having a length of less than 40 residues.
 189. The isolatedpeptide or peptidomimetic of claim 180, having a length of less than 30residues. 190-201. (canceled)